Immuno-interactive fragments of the alpha-C subunit of inhibin

ABSTRACT

Novels immuno-interactive fragments of the (alpha)C portion of a mammalian inhibin alpha subunit are disclosed, together with their variants and derivatives for producing antigen-binding molecules that are interactive with said (alpha)C portion, which are chemically well defined and which can be produced in commercially significant quantities. The antigen-binding molecules of the invention can be used for the detection of a mammalian inhibin and for the treatment and/or prevention of conditions associated with aberrant levels of a mammalian inhibin.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation of International ApplicationNo. PCT/AU00/01258, filed Oct. 18, 2000, which was published in theEnglish language on Apr. 26, 2001, under International Publication No.WO 01/29079, and the disclosure of which is incorporated herein byreference.

BACKGROUND OF THE INVENTION

[0002] The present invention relates generally to novel antigens fordeveloping antigen-binding molecules that are interactive with mammalianinhibins. More particularly, the present invention relates toimmuno-interactive fragments of the (alpha)C portion of a mammalianinhibin alpha subunit and to variants and derivatives of theseimmuno-interactive fragments for producing novel antigen-bindingmolecules that recognize the said (alpha)C portion. The invention isalso concerned with the use of these antigen-binding molecules fordetecting a mammalian inhibin and for treating or preventing conditionsassociated with aberrant levels of a mammalian inhibin.

[0003] Inhibin is a dimeric glycoprotein produced by diverse tissuesincluding the gonads, pituitary, brain, bone marrow, placenta, andadrenal gland. It was initially identified by its ability to inhibit thesecretion of follicle stimulating hormone (FSH) by the pituitary (forreviews, see Vale et al., 1990, The inhibin/activin family of hormoneand growth factors. In Peptide growth factors and their receptors:Handbook of Experimental Physiology 95:211-248 (Eds. Spom and Roberts)Springer-Verlag, Berlin; Burger, 1992, Reproductive Medicine Review1:1-20; Baird & Smith, 1993, Oxford Rev. Reprod. Biol. 15:191-232).However, it was also found subsequently to be secreted by mucinous andgranulosa cell cancers of the ovary. Thus, measurement of serum inhibinin women, particularly postmenopausal women, provides a good diagnostictest for detecting these cancers (Lapphorn et al., 1989, New England 0.1Med. 321:790-793; Healy et al., 1993, N. Eng. J. Med. 329:1539-420) andfor monitoring their recurrence after surgery. The mucinous andgranulosa cell cancers represent 20-30% of all ovarian cancers. Seruminhibin is less effective as a marker of serous cancer, which is themajor (40%) ovarian cancer. In contrast, a widely used cancer marker,CA125, is effective in the detection of serous cancers and less so withthe mucinous and granulosa cell cancers.

[0004] Inhibin consists of two chains, the alpha subunit (made up of 3regions, Pro, (alpha)N and (alpha)C) and either the (beta)A subunit(inhibin A) or (beta)B subunit (inhibin B), of varying molecular weight.Various inhibin assays with specificities directed towards differentregions of the inhibin molecule have been developed for diagnosis ofovarian cancer.

[0005] Initial studies by Lapphorn et al. (1989, supra) and Healy et al.(1993, supra) suggested that measurement of serum inhibin byradioimmunoassay (RIA) which detects (alpha)C inhibin forms may be ofdiagnostic value in monitoring mucinous and granuloma cell tumours.Whilst this method is reliable, it is less sensitive and practical incomparison to two-site or sandwich antibody assays using, for example,colorimetric or fluorescent labels for detection.

[0006] A two-site immunofluorometric assay ((alpha)C IFMA) for the(alpha)C portion of the alpha subunit of inhibin has been developed byRobertson et al. (1996, J. Clin. Endocrinol. Metab. 81:669-676). Thisassay, which utilizes sheep polyclonal antisera and the fluorescentlabel Europium (Eu), detects all known inhibin alpha subunit-containingproteins. Compared to other inhibin assays specific for the alphasubunit or the (alpha)(beta) dimers (inhibin A and B), the (alpha)C IFMAand the (alpha)C RIA have been shown to be more effective in detectingdifferent ovarian cancers (Robertson et al., 1999, Clin. Endocrinol.50:381-387; ibid, Clin. Chemistry 45:651-658).

[0007] Robertson et al. (1999, Clin. Chemistry 45:651-658) have alsoshown that 89-90% of all ovarian cancers can be detected by the (alpha)CIFMA in combination with an immunoassay for the ovarian cancer markerCA125. This combined detection value was considerably higher than foreach assay alone or a combination of CA125 with other inhibin assays,and is clinically useful in the diagnosis of the majority of ovariancancers. Furthermore, in view of its increased sensitivity, the (alpha)CIFMA is able to detect the increase in serum inhibin associated with arecurrence of granulosa cell tumours at an earlier time followingsurgery. The earlier detection of the cancer is desirable for successfultreatment.

[0008] Despite the clinical utility of the (alpha)C IFMA, the use ofpolyclonal antisera in this immunoassay or other types of multi-siteassays in the diagnostic market is a disadvantage owing to the inherentlimited supply of polyclonal antisera and the difficulties of qualitycontrol including specificity between antiserum batches. It wouldtherefore be beneficial to utilize monoclonal antisera or otherantigen-binding molecules where the stocks are potentially limitless andthe quality can be more easily monitored.

BRIEF SUMMARY OF THE INVENTION

[0009] The present invention is predicated in part on the determinationof various immuno-interactive fragments of the (alpha)C portion of aninhibin alpha subunit, which fragments interact with polyclonal antiseraraised against the (alpha)C portion. These fragments have utility inproducing antigen-binding molecules that are interactive with said(alpha)C portion, that are chemically well defined and that can beproduced in commercially significant quantities. The antigen-bindingmolecules so produced can be used for the detection of a mammalianinhibin and for the treatment and/or prevention of conditions associatedwith aberrant levels of a mammalian inhibin.

[0010] Accordingly, in one aspect of the invention, there is provided animmuno-interactive fragment of the (alpha)C portion of a mammalianinhibin alpha subunit, or variant or derivative of said fragment,wherein said fragment is interactive with a polyclonal antibody raisedagainst said (alpha)C portion.

[0011] Preferably, the polyclonal antibody is an ovine polyclonalantibody. In a preferred embodiment, the ovine polyclonal antibody isselected from the group consisting of As #41, As #128 (Robertson et al.,1996, supra) and As #1989 (Lapphorn et al., 1989, supra).

[0012] Suitably, the mammalian inhibin alpha subunit is a human inhibinalpha subunit.

[0013] The (alpha)C portion preferably comprises the sequence set forthin SEQ ID NO: 2.

[0014] Suitably, said immuno-interactive fragment comprises a sequenceselected from any one or more of SEQ ID NOs: 3, 4, 5, 6, 18, 19, 20, 21,22, 23, 30, 31, 32, 35, 36, 37, 38, 39, 40, 55, 56, 57, 58, 59, 60, 68,69, 70, 71, 72 and 73.

[0015] In one embodiment, said immuno-interactive fragment preferablycomprises a sequence selected from any one or more of SEQ ID NOs: 5, 35,36, 37, 38, 39 and 40.

[0016] In another embodiment, said immuno-interactive fragmentpreferably comprises a sequence selected from any one or more of SEQ IDNOs: 18, 19, 20, 21, 22, 23, 31, 32, 55, 56, 57, 58, 59 and 60.

[0017] In yet another embodiment, said immuno-interactive fragmentpreferably comprises a sequence selected from any one or more of SEQ IDNOs: 68, 69, 70, 71, 72 and 73.

[0018] In another aspect, the invention contemplates a method ofproducing a variant of an immuno-interactive fragment as broadlydescribed above, including the steps of:

[0019] (a) combining a compound suspected of being said variant with atleast one antigen-binding molecule that binds to said immuno-interactivefragment; and

[0020] (b) detecting the presence of a conjugate comprising saidcompound and said antigen-binding molecule, which indicates that saidcompound is a said variant.

[0021] In yet another aspect, the invention resides in anantigen-binding molecule that binds specifically to animmuno-interactive fragment of inhibin (alpha)C as broadly describedabove or variant or derivative thereof, with the proviso that saidantigen-binding molecule is other than a member selected from the groupconsisting of a polyclonal antibody and the R1 monoclonal antibodydescribed by Groome et al (1993, J. Immunol. Meth. 165:167-176; 1994,Clin. Endocrinol. 40:717-723).

[0022] In a further aspect, the invention provides a method of producingan antigen-binding molecule that binds specifically to animmuno-interactive fragment of inhibin (alpha)C as broadly describedabove or variant or derivative thereof, comprising:

[0023] (a) producing an antigen-binding molecule against inhibin(alpha)C or fragment thereof;

[0024] (b) combining the antigen-binding molecule with saidimmuno-interactive fragment, variant or derivative; and

[0025] (c) detecting the presence of a conjugate comprising saidantigen-binding molecule and said fragment.

[0026] In yet another aspect, the invention resides in the use of animmuno-interactive fragment, variant or derivative according to thepresent invention to produce an antigen-binding molecule that bindsspecifically to the (alpha)C portion of a mammalian inhibin alphasubunit and preferably to a region of said (alpha)C portioncorresponding to said immuno-interactive fragment.

[0027] In yet another aspect, the invention provides antigen-bindingmolecules so produced, with the proviso that said antigen-bindingmolecule is other than a member selected from the group consisting of apolyclonal antibody and the R1 monoclonal antibody described by Groomeet al (1993, J. Immunol. Meth. 165:167-176; 1994, Clin. Endocrinol.40:717-723).

[0028] In another aspect, the invention provides a composition for usein eliciting an immune response in a mammal which response includesproduction of elements that specifically bind the (alpha)C portion of amammalian inhibin alpha subunit, said composition comprising animmuno-interactive fragment, variant or derivative as broadly describedabove, together with a pharmaceutically acceptable carrier.

[0029] Optionally, said composition further comprises an adjuvant.

[0030] In yet another aspect of the invention there is provided a methodfor eliciting an immune response in a mammal which response includesproduction of elements that specifically bind the (alpha)C portion of amammalian inhibin alpha subunit, comprising administering to said mammalan immunogenically effective amount of a composition as broadlydescribed above.

[0031] In another aspect, the invention provides an isolatedpolynucleotide encoding an immuno-interactive fragment, variant orderivative as broadly described above.

[0032] In yet another aspect, the invention features an expressionvector comprising a polynucleotide as broadly described above whereinthe polynucleotide is operably linked to a regulatory polynucleotide.

[0033] In a further aspect, the invention provides a host cellcontaining a said expression vector.

[0034] According to another aspect of the invention, there is provided amethod of detecting a mammalian inhibin in a biological sample suspectedof containing it, comprising:

[0035] (a) contacting the biological sample with an antigen-bindingmolecule as broadly described above; and

[0036] (b) detecting the presence of a complex comprising the saidantigen-binding molecule and the mammalian inhibin in said contactedsample.

[0037] In another aspect of the invention, there is provided a method ofdiagnosing a condition associated with an aberrant concentration of amammalian inhibin in a biological sample of a patient, comprising:

[0038] (a) contacting the biological sample with an antigen-bindingmolecule as broadly described above;

[0039] (b) measuring the concentration of a complex comprising the saidantigen-binding molecule and the mammalian inhibin in said contactedsample; and

[0040] (c) relating said measured complex concentration to theconcentration of mammalian inhibin in said sample, wherein the presenceof said aberrant concentration is indicative of said condition.

[0041] Suitably, the condition is a cancer. Preferably, the cancer is ofa tissue selected from the group consisting of ovary, uterus, breast,pituitary, testis and prostate. In a preferred embodiment, the cancer isovarian cancer.

[0042] In yet another aspect, the invention contemplates a method ofdiagnosing a condition associated with an aberrant concentration of amammalian inhibin and an aberrant concentration of another antigen in abiological sample of a patient, comprising:

[0043] (a) contacting a biological sample of the patient with a firstantigen-binding molecule that binds specifically to the (alpha)C portionof a mammalian inhibin alpha subunit as broadly described above;

[0044] (b) contacting said biological sample or another biologicalsample obtained from said patient with a second antigen-binding moleculethat is immuno-interactive with said other antigen;

[0045] (c) measuring the concentration of a first complex comprising thefirst antigen-binding molecule and the mammalian inhibin in saidcontacted sample;

[0046] (d) measuring the concentration of a second complex comprisingthe second antigen-binding molecule and the other antigen in saidcontacted sample; and

[0047] (e) relating said measured complex concentrations to theconcentration of mammalian inhibin and the concentration of the otherantigen in said sample, wherein the presence of said aberrantconcentrations is indicative of said condition.

[0048] In a preferred embodiment, the condition is ovarian cancer andthe other antigen is an ovarian cancer marker. In this instance, theovarian cancer marker is preferably CA125.

[0049] In yet another aspect of the invention, there is provided amethod for treating or preventing a condition associated with anaberrant concentration of a mammalian inhibin in a mammal, comprisingadministering to said mammal a therapeutically effective amount of acomposition as broadly described above.

[0050] The invention also extends to the use of the immuno-interactivefragment, variant or derivative according to the present invention orthe use of the antigen-binding molecule mentioned above in a kit fordetecting and/or measuring mammalian inhibin in a biological sample.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

[0051] The foregoing summary, as well as the following detaileddescription of preferred embodiments of the invention, will be betterunderstood when read in conjunction with the appended drawings. For thepurpose of illustrating the invention, there is shown in the drawingsembodiments which are presently preferred. It should be understood,however, that the invention is not limited to the precise arrangementsand instrumentalities shown.

[0052] In the drawings:

[0053]FIG. 1 depicts a pair of histograms showing the binding ofantiserum (As) #41 and #128 to each of the 31 biotinylated peptidesimmobilized to streptavidin-coated surface. The binding was assessed bybinding of an enzyme-linked anti-IgG antibody. Dashed lines refer to theassay sensitivity. See text for further details. Based on these andother studies, 4 pools were formed as shown (Regions I, II, III, IV) andused in further analysis with Assays 2 and 3.

[0054]FIG. 2 is a graph showing ED₅₀ values obtained for the 31biotinylated peptides with As #41 and As #128 in the RIA format. The RIAprovides a measure of both specificity and affinity of the binding ofthe biotinylated peptides to the antisera.

[0055]FIG. 3 shows nine histograms relating to a competitive 2-siteassay. These histograms show the inhibition of inhibin binding bybiotinylated peptide pools from Regions I-IV, I, IIII, IV, peptide #5,#20 and #30 with As #128. This assay design enables the assessment ofthe epitopes identified in the various antisera in a two-antibodysandwich assay design. Legend: C, control; Bl, blank; hatched areas,peptide alone.

[0056]FIG. 4 shows ten histograms relating to a competitive 2-siteassay. These histograms show the inhibition of inhibin binding bypeptide pools from Regions I-IV, I, IIII, IV, peptide #5, #20 and #30with As #41. Legend: C, control; Bl, blank; hatched areas peptide alone.

[0057]FIG. 5 shows four histograms relating to a competitive 2-siteassay. These histograms show the inhibition of inhibin binding bypeptide #5, #20 and #29 with As #128. Legend: C, control; Bl, blank,hatched areas peptide alone.

[0058]FIG. 6 shows four histograms relating to a competitive 2-siteassay. These histograms show the inhibition of inhibin binding bypeptide #5, #20 and #29 with As #41. Legend: C, control; Bl, blank;hatched areas peptide alone.

[0059]FIG. 7 depicts three graphs showing the effect of immunoabsorptionwith peptides #5, #20 and #30 of antisera #41 and #128 in the (alpha)CIFMA. Quantitative aspects are presented in Table 5.

[0060]FIG. 8 depicts a graph showing the effect of immunoabsorption ofantiserum #41 used as both coating and labeled antibody with peptide #5in an IFMA format. In the absence of added inhibin, the blank (0 inhibindose) showed considerable binding indicating that the #5 peptide is abridge between the coated and labeled #41 antibody and thus probablycontaining two binding sites.

[0061]FIG. 9 illustrates a putative three-dimensional structure of thecarboxyl-terminal region of the inhibin alpha subunit as adapted fromthe three dimensional structure of TGF(beta). The amino acid positionsof peptides #5, #20 and #30 are presented as shaded areas.

[0062]FIG. 10A shows inhibin alpha ELISA dose response curves of inhibinA standard (1.5-100 picograms/well), various serum pools (3-50microliters/well) and human follicular fluid (hFF, XXX) using themonoclonal antibodies PO#14 and R1. Legend:

[0063]FIG. 10B shows inhibin alpha ELISA dose response curves of inhibinA standard (1.5-100 picograms/well), various serum pools (3-50microliters/well) and human follicular fluid (hFF, XXX) using themonoclonal antibodies PO#23 and R1.

[0064]FIG. 10C shows inhibin alpha ELI SA dose response curves ofinhibin A standard (1.5-100 picograms/well), various serum pools (3-50microliters/well) and human follicular fluid (hFF, XXX) using themonoclonal antibodies PO# 14, PO#23 and R1.

[0065]FIG. 11 illustrates molecular weight patterns of inhibin in serumfrom women stimulated with gonadotropins as part of an in vitrofertilization procedure (IVF serum) and male serum. The serum wasfractionated using an immunoaffinity, preparative-PAGE andelectroelution procedure (Robertson et al 1996, 1997, supra). Horizontaldashed line refers to detection limit.

[0066]FIG. 12 shows molecular weight patterns of inhibin in serum frompostmenopausal women with granulosa cell tumours and mucinous cancer.The serum was fractionated using an immunoaffinity, preparative-PAGE andelectroelution procedure (Robertson et al. 1996, 1997, supra).Horizontal dashed line refers to detection limits of the various assays.

[0067]FIG. 13 shows regression analyses of serum inhibin values fromwomen with all ovarian cancers as determined by a) IFMA and 14-R1 ELISA,b) IFMA and 23-R1 ELISA, c) IFMA and 14+23-R1 ELISA, and d) 23-R1 ELISAand 14-R1 ELISA. Dashed lines refer to the detection limits of thevarious assays.

DETAILED DESCRIPTION OF THE INVENTION

[0068] 1. Definitions

[0069] Unless defined otherwise, all technical and scientific terms usedherein have the same meaning as commonly understood by those of ordinaryskill in the art to which the invention belongs. Although any methodsand materials similar or equivalent to those described herein can beused in the practice or testing of the present invention, preferredmethods and materials are described. For the purposes of the presentinvention, the following terms are defined below.

[0070] The articles “a” and “an” are used herein to refer to one or tomore than one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

[0071] “Amplification product” refers to a nucleic acid productgenerated by nucleic acid amplification techniques.

[0072] By “antigen-binding molecule” is meant a molecule that hasbinding affinity for a target antigen. It will be understood that thisterm extends to immunoglobulins, immunoglobulin fragments andnon-immunoglobulin derived protein frameworks that exhibitantigen-binding activity.

[0073] The term “biological sample” as used herein refers to a samplethat may be extracted, untreated, treated, diluted or concentrated froma patient. The biological sample may be selected from the groupconsisting of whole blood, serum, plasma, saliva, urine, sweat, asciticfluid, peritoneal fluid, synovial fluid, amniotic fluid, cerebrospinalfluid, skin biopsy, and the like. The biological sample preferablyincludes serum, whole blood, plasma, lymph and ovarian follicular fluidas well as other circulatory fluid and saliva, mucus secretion andrespiratory fluid. More preferably, the biological sample is acirculatory fluid such as serum or whole blood or a fractionated portionthereof. Most preferably, the biological sample is serum or afractionated portion thereof.

[0074] By “condition associated with an aberrant concentration” is meantany condition including a healthy condition or an unhealthy conditionthat is associated with a concentration of the (alpha)C portion of amammalian inhibin alpha subunit which concentration deviatessignificantly from a corresponding normal concentration range. Suitably,the condition is a cancer including ovarian, prostate, testicular,pituitary, breast and uterine cancer.

[0075] By “corresponds to” or “corresponding to” is meant apolynucleotide (a) having a nucleotide sequence that is substantiallyidentical or complementary to all or a portion of a referencepolynucleotide sequence or (b) encoding an amino acid sequence identicalto an amino acid sequence in a peptide or protein. This phrase alsoincludes within its scope a peptide or polypeptide having an amino acidsequence that is substantially identical to a sequence of amino acids ina reference peptide or protein.

[0076] By “derivative” is meant a polypeptide that has been derived fromthe basic sequence by modification, for example by conjugation orcomplexing with other chemical moieties or by post-translationalmodification techniques as would be understood in the art. The term“derivative” also includes within its scope alterations that have beenmade to a parent sequence including additions, or deletions that providefor functional equivalent molecules. Accordingly, the term derivativeencompasses molecules that will elicit an immune response against the(alpha)C portion of a mammalian inhibin alpha subunit.

[0077] For the purposes of the present invention, the phrase “elicit(s)an immune response” refers to the ability of the aforementionedimmuno-interactive fragment or variant to produce an immune response ina mammal to which it is administered, wherein the response includes theproduction of elements which specifically bind the (alpha)C portion of amammalian inhibin alpha subunit.

[0078] “Homology” refers to the percentage number of amino acids thatare identical or constitute conservative substitutions as defined inTable A below. Homology may be determined using sequence comparisonprograms such as GAP (Deveraux et al., 1984, Nucl. Acids Res.12:387-395). In this way, sequences of a similar or substantiallydifferent length to those cited herein might be compared by insertion ofgaps into the alignment, such gaps being determined, for example, by thecomparison algorithm used by GAP.

[0079] “Hybridization” is used herein to denote the pairing ofcomplementary nucleotide sequences to produce a DNA-DNA hybrid or aDNA-RNA hybrid. Complementary base sequences are those sequences thatare related by the base-pairing rules. In DNA, A pairs with T and Cpairs with G. In RNA U pairs with A and C pairs with G. In this regard,the terms “match” and “mismatch” as used herein refer to thehybridization potential of paired nucleotides in complementary nucleicacid strands. Matched nucleotides hybridize efficiently, such as theclassical A-T and G-C base pair mentioned above. Mismatches are othercombinations of nucleotides that do not hybridize efficiently.

[0080] By “immunologically effective amount” is meant the administrationto a mammal of an amount of an immuno-interactive fragment, variant orderivative of the invention, either in a single dose or as part of aseries, that is effective for raising an immune response against the(alpha)C portion of a mammalian inhibin alpha subunit. The effectiveamount will vary depending upon the taxonomic group of mammal to betreated, the capacity of the individual's immune system to elicit animmune response (inclusive of a humoral and/or a cellular immuneresponse), the formulation of the vaccine. It is expected that theamount will fall in a relatively broad range that can be determinedthrough routine trials.

[0081] Reference herein to “immuno-interactive” includes reference toany interaction, reaction, or other form of association betweenmolecules and in particular where one of the molecules is, or mimics, acomponent of the immune system.

[0082] By “immuno-interactive fragment” is meant a fragment of the(alpha)C portion of a mammalian inhibin alpha subunit which fragmentelicits an immune response against the said alpha subunit, andpreferably against a human inhibin alpha subunit. For example, in thecase of an immuno-interactive fragment according to any one of SEQ IDNO: 3, 4, 5, 6, 21, 22, 23, 30, 31, 32, 35, 36, 37, 38, 39, 40, 55, 56,57, 58, 59, 60, 68, 69, 70, 71, 72 and 73, the said fragment must elicitan immune response that includes the production of elements thatspecifically bind the (alpha)C portion of a mammalian inhibin alphasubunit. As used herein, the term “immuno-interactive fragment” includesdeletion mutants and small peptides, for example of at least six,preferably at least 8 and more preferably at least 20 contiguous aminoacids, which comprise antigenic determinants or epitopes. Several suchfragments may be joined together. Peptides of this type may be obtainedthrough the application of standard recombinant nucleic acid techniquesor synthesized using conventional liquid or solid phase synthesistechniques. For example, reference may be made to solution synthesis orsolid phase synthesis as described, for example, in Chapter 9 entitled“Peptide Synthesis” by Atherton and Shephard which is included in apublication entitled “Synthetic Vaccines” edited by Nicholson andpublished by Blackwell Scientific Publications. Alternatively, peptidescan be produced by digestion of a polypeptide of the invention withproteinases such as endoLys-C, endoArg-C, endoGlu-C and staphylococcusV8-protease. The digested fragments can be purified by, for example,high performance liquid chromatographic (HPLC) techniques.

[0083] Reference herein to “inhibin” includes all forms of the moleculeincluding its precursor forms. For example, the term “inhibin” includesinhibin A, inhibin B, free inhibin alpha subunit, Pro(alpha)N(alpha)C,Pro(alpha)C and (alpha)C. Dimeric and monomeric forms of inhibin arecontemplated by the present invention. Furthermore, use of the term“inhibin” is not to impart any functional limitation on the moleculesince subunits such as Pro(alpha)C or Pro(alpha)N(alpha)C may not haveinhibin-like properties but are yet still useful in assays according tothe present invention. Most preferably, the assays of the inventiondetect the inhibin alpha subunit and reference herein to “inhibin”includes, in a preferred embodiment, the alpha subunit alone or avariant or derivative thereof including, but not limited to, Pro(alpha)Cand (alpha)C.

[0084] By “isolated” is meant material that is substantially oressentially free from components that normally accompany it in itsnative state. For example, an “isolated polynucleotide”, as used herein,refers to a polynucleotide, which has been purified from the sequenceswhich flank it in a naturally occurring state, e.g., a DNA fragmentwhich has been removed from the sequences which are normally adjacent tothe fragment.

[0085] By “obtained from” is meant that a sample such as, for example, anucleic acid extract is isolated from, or derived from, a particularsource of the host. For example, the nucleic acid extract may beobtained from tissue isolated directly from the host.

[0086] The term “oligonucleotide” as used herein refers to a polymercomposed of a multiplicity of nucleotide units (deoxyribonucleotides orribonucleotides, or related structural variants or synthetic analoguesthereof) linked via phosphodiester bonds (or related structural variantsor synthetic analogues thereof). Thus, while the term “oligonucleotide”typically refers to a nucleotide polymer in which the nucleotides andlinkages between them are naturally occurring, it will be understoodthat the term also includes within its scope various analoguesincluding, but not restricted to, peptide nucleic acids (PNAs),phosphoramidates, phosphorothioates, methyl phosphonates, 2-O-methylribonucleic acids, and the like. The exact size of the molecule may varydepending on the particular application. An oligonucleotide is typicallyrather short in length, generally from about 10 to 30 nucleotides, butthe term can refer to molecules of any length, although the term“polynucleotide” or “nucleic acid” is typically used for largeoligonucleotides.

[0087] By “operably linked” is meant that transcriptional andtranslational regulatory nucleic acids are positioned relative to apolypeptide-encoding polynucleotide in such a manner that thepolynucleotide is transcribed and the polypeptide is translated.

[0088] The term “ovarian cancer” as used herein includes collectivelyall the major forms of the disease such as forms classified as serous,mucinous, granulosa cell tumor and miscellaneous as well as cancersrelated to ovarian cancer.

[0089] The term “patient” refers to patients of human or other mammaland includes any individual it is desired to examine or treat using themethods of the invention. However, it will be understood that “patient”does not imply that symptoms are present. Suitable mammals that fallwithin the scope of the invention include, but are not restricted to,primates, livestock animals (e.g., sheep, cows, horses, donkeys, pigs),laboratory test animals (e.g., rabbits, mice, rats, guinea pigs,hamsters), companion animals (e.g., cats, dogs) and captive wild animals(e.g., foxes, deer, dingoes).

[0090] By “pharmaceutically-acceptable carrier” is meant a solid orliquid filler, diluent or encapsulating substance that may be safelyused in topical or systemic administration.

[0091] The term “polynucleotide” or “nucleic acid” as used hereindesignates mRNA, RNA, cRNA, cDNA or DNA. The term typically refers tooligonucleotides greater than 30 nucleotides in length.

[0092] The terms “polynucleotide variant” refer to polynucleotidesdisplaying substantial sequence identity with a reference polynucleotidesequence or polynucleotides that hybridize with a reference sequenceunder stringent conditions that are defined hereinafter. These termsalso encompasses polynucleotides in which one or more nucleotides havebeen added or deleted, or replaced with different nucleotides. In thisregard, it is well understood in the art that certain alterationsinclusive of mutations, additions, deletions and substitutions can bemade to a reference polynucleotide whereby the altered polynucleotideretains the biological function or activity of the referencepolynucleotide. The terms “polynucleotide sequence variant” and“variant” also include naturally occurring allelic variants.

[0093] “Polypeptide”, “peptide” and “protein” are used interchangeablyherein to refer to a polymer of amino acid residues and to variants andsynthetic analogues of the same. Thus, these terms apply to amino acidpolymers in which one or more amino acid residues is a syntheticnon-naturally occurring amino acid, such as a chemical analogue of acorresponding naturally occurring amino acid, as well as tonaturally-occurring amino acid polymers.

[0094] The term “polypeptide variant” refers to polypeptides in whichone or more amino acids have been replaced by different amino acids. Itis well understood in the art that some amino acids may be changed toothers with broadly similar properties without changing the nature ofthe activity of the polypeptide (conservative substitutions) asdescribed hereinafter. Accordingly, polypeptide variants as used hereinencompass polypeptides that will elicit an immune response against the(alpha)C portion of a mammalian inhibin alpha subunit.

[0095] By “primer” is meant an oligonucleotide which, when paired with astrand of DNA, is capable of initiating the synthesis of a primerextension product in the presence of a suitable polymerizing agent. Theprimer is preferably single-stranded for maximum efficiency inamplification but may alternatively be double-stranded. A primer must besufficiently long to prime the synthesis of extension products in thepresence of the polymerization agent. The length of the primer dependson many factors, including application, temperature to be employed,template reaction conditions, other reagents, and source of primers. Forexample, depending on the complexity of the target sequence, theoligonucleotide primer typically contains 15 to 35 or more nucleotides,although it may contain fewer nucleotides. Primers can be largepolynucleotides, such as from about 200 nucleotides to several kilobasesor more. Primers may be selected to be “substantially complementary” tothe sequence on the template to which it is designed to hybridize andserve as a site for the initiation of synthesis. By “substantiallycomplementary”, it is meant that the primer is sufficientlycomplementary to hybridize with a target nucleotide sequence.Preferably, the primer contains no mismatches with the template to whichit is designed to hybridize but this is not essential. For example,non-complementary nucleotides may be attached to the 5′-end of theprimer, with the remainder of the primer sequence being complementary tothe template. Alternatively, non-complementary nucleotides or a stretchof non-complementary nucleotides can be interspersed into a primer,provided that the primer sequence has sufficient complementarity withthe sequence of the template to hybridize therewith and thereby form atemplate for synthesis of the extension product of the primer.

[0096] “Probe” refers to a molecule that binds to a specific sequence orsub-sequence or other moiety of another molecule. Unless otherwiseindicated, the term “probe” typically refers to a polynucleotide probethat binds to another nucleic acid, often called the “target nucleicacid”, through complementary base pairing. Probes may bind targetnucleic acids lacking complete sequence complementarity with the probe,depending on the stringency of the hybridization conditions. Probes canbe labeled directly or indirectly.

[0097] The term “recombinant polynucleotide” as used herein refers to apolynucleotide formed in vitro by the manipulation of nucleic acid intoa form not normally found in nature. For example, the recombinantpolynucleotide may be in the form of an expression vector. Generally,such expression vectors include transcriptional and translationalregulatory nucleic acid operably linked to the nucleotide sequence.

[0098] By “recombinant polypeptide” is meant a polypeptide made usingrecombinant techniques, i.e., through the expression of a recombinantpolynucleotide.

[0099] By “reporter molecule” as used in the present specification ismeant a molecule that, by its chemical nature, provides an analyticallyidentifiable signal that allows the detection of a complex comprising anantigen-binding molecule and its target antigen. The term “reportermolecule” also extends to use of cell agglutination or inhibition ofagglutination such as red blood cells on latex beads, and the like.

[0100] Terms used to describe sequence relationships between two or morepolynucleotides or polypeptides include “reference sequence”,“comparison window”, “sequence identity”, “percentage of sequenceidentity” and “substantial identity”. A “reference sequence” is at least6 but frequently 15 to 18 and often at least 25 monomer units, inclusiveof nucleotides and amino acid residues, in length. Because twopolynucleotides may each comprise (1) a sequence (i.e., only a portionof the complete polynucleotide sequence) that is similar between the twopolynucleotides, and (2) a sequence that is divergent between the twopolynucleotides, sequence comparisons between two (or more)polynucleotides are typically performed by comparing sequences of thetwo polynucleotides over a “comparison window” to identify and comparelocal regions of sequence similarity. A “comparison window” refers to aconceptual segment of typically 12 contiguous residues that is comparedto a reference sequence. The comparison window may comprise additions ordeletions (i.e., gaps) of about 20% or less as compared to the referencesequence (which does not comprise additions or deletions) for optimalalignment of the two sequences. Optimal alignment of sequences foraligning a comparison window may be conducted by computerizedimplementations of algorithms (GAP, BESTFIT, FASTA, and TFASTA in theWisconsin Genetics Software Package Release 7.0, Genetics ComputerGroup, 575 Science Drive Madison, Wis., USA) or by inspection and thebest alignment (i.e., resulting in the highest percentage homology overthe comparison window) generated by any of the various methods selected.Reference also may be made to the BLAST family of programs as forexample disclosed by Altschul et al., 1997, Nucl. Acids Res. 25:3389. Adetailed discussion of sequence analysis can be found in Unit 19.3 ofAusubel et al., “Current Protocols in Molecular Biology”, John Wiley &Sons Inc, 1994-1998, Chapter 15.

[0101] The term “sequence identity” as used herein refers to the extentthat sequences are identical on a nucleotide-by-nucleotide basis or anamino acid-by-amino acid basis over a window of comparison. Thus, a“percentage of sequence identity” is calculated by comparing twooptimally aligned sequences over the window of comparison, determiningthe number of positions at which the identical nucleic acid base (e.g.,A, T, C, G, I) or the identical amino acid residue (e.g., Ala, Pro, Ser,Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn,Gln, Cys and Met) occurs in both sequences to yield the number ofmatched positions, dividing the number of matched positions by the totalnumber of positions in the window of comparison (i.e., the window size),and multiplying the result by 100 to yield the percentage of sequenceidentity. For the purposes of the present invention, “sequence identity”will be understood to mean the “match percentage” calculated by theDNASIS computer program (Version 2.5 for windows; available from HitachiSoftware engineering Co., Ltd., South San Francisco, Calif., USA) usingstandard defaults as used in the reference manual accompanying thesoftware.

[0102] “Stringency” as used herein, refers to the temperature and ionicstrength conditions, and presence or absence of certain organicsolvents, during hybridization. The higher the stringency, the higherwill be the degree of complementarity between immobilized nucleotidesequences and the labeled polynucleotide sequence.

[0103] “Stringent conditions” refers to temperature and ionic conditionsunder which only nucleotide sequences having a high frequency ofcomplementary bases will hybridize. The stringency required isnucleotide sequence dependent and depends upon the various componentspresent during hybridization. Generally, stringent conditions areselected to be about 10 to 20° C. lower than the thermal melting point(T_(m)) for the specific sequence at a defined ionic strength and pH.The T_(m) is the temperature (under defined ionic strength and pH) atwhich 50% of a target sequence hybridizes to a complementary probe.

[0104] The term “substantially pure” as used herein describes acompound, e.g., a peptide that has been separated from components thatnaturally accompany it. Typically, a compound is substantially pure whenat least 60%, more preferably at least 75%, more preferably at least90%, and most preferably at least 99% of the total material (by volume,by wet or dry weight, or by mole percent or mole fraction) in a sampleis the compound of interest. Purity can be measured by any appropriatemethod, e.g., in the case of polypeptides, by chromatography, gelelectrophoresis or HPLC analysis. A compound, e.g., a polypeptide isalso substantially purified when it is essentially free of naturallyassociated components when it is separated from the native contaminantswhich accompany it in its natural state.

[0105] By “vector” is meant a nucleic acid molecule, preferably a DNAmolecule derived, for example, from a plasmid, bacteriophage, or plantvirus, into which a nucleic acid sequence may be inserted or cloned. Avector preferably contains one or more unique restriction sites and maybe capable of autonomous replication in a defined host cell including atarget cell or tissue or a progenitor cell or tissue thereof, or beintegrable with the genome of the defined host such that the clonedsequence is reproducible. Accordingly, the vector may be an autonomouslyreplicating vector, i.e., a vector that exists as an extrachromosomalentity, the replication of which is independent of chromosomalreplication, e.g., a linear or closed circular plasmid, anextrachromosomal element, a minichromosome, or an artificial chromosome.The vector may contain any means for assuring self-replication.Alternatively, the vector may be one which, when introduced into thehost cell, is integrated into the genome and replicated together withthe chromosome(s) into which it has been integrated. A vector system maycomprise a single vector or plasmid, two or more vectors or plasmids,which together contain the total DNA to be introduced into the genome ofthe host cell, or a transposon. The choice of the vector will typicallydepend on the compatibility of the vector with the host cell into whichthe vector is to be introduced. The vector may also include a selectionmarker such as an antibiotic resistance gene that can be used forselection of suitable transformants. Examples of such resistance genesare well known to those of skill in the art.

[0106] Throughout this specification and the appended claims, unless thecontext requires otherwise, the words “comprise”, “comprises” and“comprising” will be understood to imply the inclusion of a statedinteger or step or group of integers or steps but not the exclusion ofany other integer or step or group of integers or steps.

[0107] 2. Immuno-Interactive Molecules of the Invention

[0108] 2.1. Immuno-Interactive Fragments of the (alpha)C Portion of aMammalian Inhibin Alpha Subunit

[0109] The present invention provides an immuno-interactive fragment ofthe (alpha)C portion of a mammalian inhibin alpha subunit, whichfragment is interactive with a polyclonal antiserum raised against thesaid (alpha)C portion. Preferably, the polyclonal antiserum is an ovinepolyclonal antiserum as for example obtained by the method by Robertsonet al. (1997, J. Clin. Endocrinol. Metabol. 82:889-896).

[0110] Suitably, the mammalian inhibin alpha subunit is a human inhibinalpha subunit. Accordingly, the said (alpha)C portion preferablycomprises the sequence set forth in SEQ ID NO: 2. SEQ ID NO: 2 encodesthe (alpha)C portion of human inhibin alpha subunit and corresponds to a134-amino acid residue fragment of human inhibin alpha subunit, spanningresidue 233 through residue 366 of the inhibin alpha subunit precursoras for example disclosed under Accession No. AAA59166 of the GenPeptdatabase (National Center for Biotechnology Information).

[0111] In a preferred embodiment, the immuno-interactive fragmentcomprises the sequence set forth in any one or more of SEQ ID NO: 3, 4,5, 6, 18, 19, 20, 21, 22, 23, 30, 31, 32, 35, 36, 37, 38, 39, 40, 55,56, 57, 58, 59, 60, 68, 69, 70, 71, 72 and 73. The correspondingpositions of these immuno-interactive fragments relative to the aminoacid sequence of the (alpha)C portion of human inhibin alpha subunit(set forth in SEQ ID NO: 2) are presented in Tables 1 and 7 infra.

[0112]2.2. Identification of Immuno-Interactive Fragments

[0113] Immuno-interactive fragments may be identified according to anysuitable procedure known in the art. For example, a suitable method mayinclude generating a fragment of a polypeptide according to any one ormore of SEQ ID NO: 3, 4, 5, 6, 18, 19, 20, 21, 22, 23, 30, 31, 32, 35,36, 37, 38, 39, 40, 55, 56, 57, 58, 59, 60, 68, 69, 70, 71, 72 and 73,administering the fragment to a mammal, and detecting an immune responsein the mammal. Such response will include production of elements thatspecifically bind the (alpha)C portion of a mammalian inhibin alphasubunit, preferably the (alpha)C portion of human inhibin alpha subunit.

[0114] Prior to testing a particular fragment for immunoreactivity inthe above method, a variety of predictive methods may be used to deducewhether a particular fragment can be used to obtain an antibody thatcross-reacts with the native antigen. These predictive methods may bebased on amino-terminal or carboxyl-terminal sequences as for exampledescribed in Chapter 11.14 of Ausubel et al., (1994-1998, supra).Alternatively, these predictive methods may be based on predictions ofhydrophilicity as for example described by Kyte and Doolittle (1982, J.Mol. Biol. 157:105-132) and Hopp and Woods (1983, Mol. Immunol.20:483-489), or predictions of secondary structure as for exampledescribed by Choo and Fasman (1978, Ann. Rev. Biochem. 47:251-276).

[0115] Generally, peptide fragments consisting of 10 to 15 residuesprovide optimal results. Peptides as small as 6 or as large as 20residues have worked successfully. Such peptide fragments may then bechemically coupled to a carrier molecule such as keyhole limpethemocyanin (KLH) or bovine serum albumin (BSA) as for example describedin Chapters 11.14 and 11.15 of Ausubel et al., (1994-1998, supra).

[0116] The peptides may be used to immunize a mammal as for examplediscussed above. Antibody titers against the native or parentpolypeptide from which the peptide was selected may then be determinedby radioimmunoassay or ELISA as for instance described in Chapters 11.16and 114 of Ausubel et al., (1994-1998, supra).

[0117] Antibodies may then be purified from a suitable biological fluidof the animal by ammonium sulfate fractionation or by chromatography asis well known in the art. Exemplary protocols for antibody purificationis given in Chapters 10.11 and 11.13 of Ausubel et al., (1994-1998,supra). Immunoreactivity of the antibody against the native or parentpolypeptide may be determined by any suitable procedure such as, forexample, western blot.

[0118] Polypeptide Variants

[0119] The invention also contemplates polypeptide variants of theimmuno-interactive fragment of the invention wherein said variantselicit an immune response against the (alpha)C portion of a mammalianinhibin alpha subunit the (alpha)C portion of a mammalian inhibin alphasubunit. In general, variants will be at least 75% homologous, moresuitably at least 80%, preferably at least 85%, and more preferably atleast 90% homologous to an immuno-interactive fragment as for exampleshown in SEQ ID NO: 3, 4, 5, 6, 18, 19, 20, 21, 22, 23, 30, 31, 32, 35,36, 37, 38, 39, 40, 55, 56, 57, 58, 59, 60, 68, 69, 70, 71, 72 and 73.It is preferred that variants display at least 60%, more suitably atleast 70%, preferably at least 75%, more preferably at least 80%, morepreferably at least 85%, more preferably at least 90% and still morepreferably at least 95% sequence identity with an immuno-interactivefragment as for example shown in SEQ ID NO: 3, 4, 5, 6, 18, 19, 20, 21,22, 23, 30, 31, 32, 35, 36, 37, 38, 39, 40, 55, 56, 57, 58, 59, 60, 68,69, 70, 71, 72 and 73. In this respect, the window of comparisonpreferably spans about the full length of the immuno-interactivefragment.

[0120] Suitably, the polypeptide variants of the invention willcross-react with or mimic immunologically an epitope of the (alpha)Cportion of a mammalian inhibin alpha subunit. Thus, polypeptide variantsaccording to the invention may bind an antigen-binding molecule thatalso binds an epitope of the (alpha)C portion of a mammalian inhibinalpha subunit and preferably the (alpha)C portion of a human inhibinalpha subunit.

[0121] Suitable polypeptide variants may be identified by combining acompound suspected of being a variant with at least one antigen-bindingmolecule that binds to the said (alpha)C portion. If a conjugate isformed comprising the compound and the antigen-binding molecule, this isindicative of the compound being a variant of the aforementionedimmuno-interactive fragment. In a preferred embodiment, the compound ispreferably a polypeptide (e.g., a modified polypeptide) whose sequenceis distinguished from the immuno-interactive fragment by substitution,deletion and/or addition of at least one amino acid.

[0122] 2.3.1. Assay Formats for Detecting Polypeptide Variants

[0123] Any suitable technique for determining formation of the conjugatemay be used. For example, the antigen-binding molecule may be utilizedin conventional immunoassays. Such immunoassays may include, but are notlimited to, radioimmunoassays (RIAs), enzyme-linked immunosorbent assays(ELISAs) and immunochromatographic techniques (ICTs) which are wellknown those of skill in the art. For example, reference may be made toColigan et al. (“Current Protocols in Immunology”, John Wiley & Sons,Inc, 1995-1997), in which a variety of immunoassays are described thatmay be used in accordance with the present invention. In this regard,the invention contemplates any immunoassay that can detect the presenceof a conjugate as herein described. For example, immunoassays mayinclude competitive and non-competitive assays as understood in the art.Such immunoassays may be carried out in solution or, at least in part,on solid supports, e.g., microtiter plates, polystyrene beads,nitrocellulose membranes, glass fiber membranes, immunochromatographicstrips, and the like. The two most common formats for immunoassays arecompetitive and non-competitive (sandwich) formats.

[0124] In a competitive format, an antigen-binding molecule such as apolyclonal or monoclonal antibody is bound to a solid support. Thisantibody is suitably capable of binding a polypeptide according to SEQID NO: 2 or immuno-interactive fragment thereof. A solution of antigenlabeled to permit detection (e.g., a labeled polypeptide orimmuno-interactive fragment) is allowed to compete with unlabelledantigen (e.g., a compound suspected of being a variant) for the solidphase antibody. The extent to which the labeled antigen is bound to thesolid phase or is detected in the solution phase can be used as ameasure of the presence of said conjugate.

[0125] In a non-competitive, or sandwich format, a polyclonal orpreferably a monoclonal antibody is bound to a solid support. Suchantibody is suitably capable of binding a polypeptide according to SEQID NO: 2 or immuno-interactive fragment thereof. In the case of apolyclonal antibody bound to the solid support, the sample containingthe suspected antigen (i.e., a compound suspected of being said variant)is allowed to contact the solid phase in order for the antigen to bindto the antibody on the solid phase. Typically, after an incubation step,the sample is separated from the solid phase, which is then washed andincubated in the presence of additional polyclonal antibody that hasbeen labeled to permit detection. Subsequently, the unbound labeledantibody is separated from the solid phase and the amount of labeledantibody in either the solution phase or bound to the solid phase in anantibody:antigen:antibody sandwich is determined as a measure of thepresence of said conjugate. In the case of a non-competitive formatemploying monoclonal antibodies, a pair of monoclonal antibodies istypically utilized, one bound to the solid support and the other labeledto permit detection. The use of monoclonal antibody pairs that recognizedifferent epitopic sites on an antigen makes it possible to conductsimultaneous immunometric assays in which the antigen and labeledantibody incubations do not require the intermediate steps of priorprocesses.

[0126] Alternatively, solid phase detection of the conjugate may bedetermined by immunoaffinity chromatography, as for example described byColigan et al., (supra, in particular Chapter 9.5) and Ausubel et al.(“Current Protocols in Molecular Biology”, John Wiley & Sons Inc,1994-1998, in particular Chapter 10.11), by immunoblotting, as forexample described by Ausubel et al. (supra, in Chapter 10.8), or byimmunoprecipitation, as for example described by Ausubel et al. (supra,in Chapter 10.16).

[0127] Solution-phase immunoassays are also contemplated by the presentinvention. For instance, detection of said conjugate may be carried outin solution using flow cytometric analysis as for example described inShapiro (“Practical Flow Cytometry”, 3rd ed., Wiley-Liss, New York,1995).

[0128] 2.3.2. Methods of Producing Polypeptide Variants

[0129] 2.3.2.1. Mutagenesis

[0130] Polypeptide variants according to the invention can be identifiedeither rationally, or via established methods of mutagenesis (see, forexample, Watson et al., “Molecular Biology of the Gene”, Fourth Edition,Benjamin/Cummings, Menlo Park, Calif., 1987). Significantly, a randommutagenesis approach requires no a priori information about the genesequence that is to be mutated. This approach has the advantage that itassesses the desirability of a particular mutant based on its function,and thus does not require an understanding of how or why the resultantmutant protein has adopted a particular conformation. Indeed, the randommutation of target gene sequences has been one approach used to obtainmutant proteins having desired characteristics (Leatherbarrow, 1986, J.Prot. Eng. 1:7-16; Knowles, 1987, Science 236:1252-1258; Shaw, 1987,Biochem. J. 246:1-17; Gerit, 1987, Chem. Rev. 87:1079-1105).Alternatively, where a particular sequence alteration is desired,methods of site-directed mutagenesis can be employed. Thus, such methodsmay be used to selectively alter only those amino acids of the proteinthat are believed to be important (Craik, 1985, Science 228:291-297;Cronin, et al., 1988, Biochemistry 27:4572-4579; Wilks, et al., 1988,Science 242:1541-1544).

[0131] Variant peptides or polypeptides, resulting from rational orestablished methods of mutagenesis or from combinatorial chemistries ashereinafter described, may comprise conservative amino acidsubstitutions. Exemplary conservative substitutions in animmuno-interactive polypeptide or polypeptide fragment according to theinvention may be made according to the following table: TABLE A OriginalResidue Exemplary Substitutions Ala Ser Arg Lys Asn Gln, His Asp Glu CysSer Gln Asn Glu Asp Gly Pro His Asn, Gln Ile Leu, Val Leu Ile, Val LysArg, Gln, Glu Met Leu, Ile, Phe Met, Leu, Tyr Ser Thr Thr Ser Trp TyrTyr Trp, Phe Val Ile, Leu

[0132] Substantial changes in function are made by selectingsubstitutions that are less conservative than those shown in Table A.Other replacements would be non-conservative substitutions andrelatively fewer of these may be tolerated. Generally, the substitutionswhich are likely to produce the greatest changes in a polypeptide'sproperties are those in which (a) a hydrophilic residue (e.g., Ser orThr) is substituted for, or by, a hydrophobic residue (e.g., Ala, Leu,Ile, Phe or Val); (b) a cysteine or proline is substituted for, or by,any other residue; (c) a residue having an electropositive side chain(e.g., Arg, His or Lys) is substituted for, or by, an electronegativeresidue (e.g., Glu or Asp) or (d) a residue having a bulky side chain(e.g., Phe or Trp) is substituted for, or by, one having a smaller sidechain (e.g., Ala, Ser)or no side chain (e.g., Gly).

[0133] What constitutes suitable variants may be determined byconventional techniques. For example, nucleic acids encoding apolypeptide according to any one or more of SEQ ID NO: 3, 4, 5, 6, 18,19, 20, 21, 22, 23, 30, 31, 32, 35, 36, 37, 38, 39, 40, 55, 56, 57, 58,59, 60, 68, 69, 70, 71, 72 and 73 can be mutated using either randommutagenesis for example using transposon mutagenesis, or site-directedmutagenesis as described, for example, in Section 3.2 herein.

[0134]2.3.2.2. Peptide Libraries Produced by Combinatorial Chemistry

[0135] A number of facile combinatorial technologies can be utilized tosynthesize molecular libraries of immense diversity. In the presentcase, variants of an immuno-interactive polypeptide, preferably animmuno-interactive polypeptide fragment according to the invention, canbe synthesized using such technologies. Variants can be screenedsubsequently using the methods described in Section 2.3.1.

[0136] Preferably, soluble synthetic peptide combinatorial libraries(SPCLs) are produced which offer the advantage of working with freepeptides in solution, thus permitting adjustment of peptideconcentration to accommodate a particular assay system. SPCLs aresuitably prepared as hexamers. In this regard, a majority of bindingsites is known to involve four to six residues. Cysteine is preferablyexcluded from the mixture positions to avoid the formation of disulfidesand more difficult-to-define polymers. Exemplary methods of producingSPCLs are disclosed by Houghten et al. (1991, Nature 354:84-86; 1992,BioTechniques 13:412-421), Appel et al. (1992, Immunomethods 1:17-23),and Pinilla et al. (1992, BioTechniques 13:901-905; 1993, Gene128:71-76).

[0137] Preparation of combinatorial synthetic peptide libraries mayemploy either t-butyloxycarbonyl (t-Boc) or 9-fluorenylmethyloxycarbonyl(Fmoc) chemistries (see Chapter 9.1, of Coligan et al., supra; Stewartand Young, 1984, Solid Phase Peptide Synthesis, 2nd ed. Pierce ChemicalCo., Rockford, Ill; and Atherton and Sheppard, 1989, Solid Phase PeptideSynthesis: A Practical Approach. IRL Press, Oxford) preferably, but notexclusively, using one of two different approaches. The first of theseapproaches, suitably termed the “split-process-recombine” or “splitsynthesis” method, was described first by Furka et al. (1988, 14th Int.Congr. Biochem., Prague, Czechoslovakia 5:47; 1991, Int. J. Pept.Protein Res. 37:487-493) and Lam et al. (1991, Nature 354:82-84), andreviewed later by Eichler et al. (1995, Medicinal Research Reviews15(6):481-496) and Balkenhohl et al. (1996, Angew. Chem. Int. Ed. Engl.35:2288-2337). Briefly, the split synthesis method involves dividing aplurality of solid supports such as polymer beads into n equal fractionsrepresentative of the number of available amino acids for each step ofthe synthesis (e.g., 20 L-amino acids), coupling a single respectiveamino acid to each polymer bead of a corresponding fraction, and thenthoroughly mixing the polymer beads of all the fractions together. Thisprocess is repeated for a total of x cycles to produce a stochasticcollection of up to Nx different compounds. The peptide library soproduced may be screened with a suitably labeled monoclonal antibody.Upon detection, some of the positive beads are selected for sequencingto identify the active peptide. Such peptide may be subsequently cleavedfrom the beads, and assayed using the same antibody to identify the mostactive peptide sequence.

[0138] The second approach, the chemical ratio method, prepares mixedpeptide resins using a specific ratio of amino acids empirically definedto give equimolar incorporation of each amino acid at each couplingstep. Each resin bead contains a mixture of peptides. Approximateequimolar representation can be confirmed by amino acid analysis (Dooleyand Houghten, 1993, Proc. Natl. Acad. Sci. USA 90:10811-10815; Eichlerand Houghten, 1993, Biochemistry 32:11035-11041). Preferably, thesynthetic peptide library is produced on polyethylene rods, or pins, asa solid support, as for example disclosed by Geysen et al. (1986, Mol.Immunol. 23:709-715). An exemplary peptide library of this type mayconsist of octapeptides in which the third and fourth position aredefined with each of the 20 amino acids, whereas the remaining sixpositions are present as mixtures. This peptide library can berepresented by the formula Ac-XXO₁O₂XXXX-S_(S), where S_(S) is the solidsupport. Peptide mixtures remain on the pins when assayed against asoluble receptor molecule. For example, the peptide library of Geysen(1986, Immunol. Today 6:364-369; and Geysen et al., Ibid), comprisingfor example dipeptides, is first screened for the ability to bind to atarget molecule. The most active dipeptides are then selected for anadditional round of testing comprising linking, to the startingdipeptide, an additional residue (or by internally modifying thecomponents of the original starting dipeptide) and then screening thisset of candidates for the desired activity. This process is reiterateduntil the binding partner having the desired properties is identified.

[0139] 2.3.2.3. Alanine Scanning Mutagenesis

[0140] In one embodiment, the invention herein utilizes a systematicanalysis of an immuno-interactive fragment according to the invention todetermine the residues in the said (alpha)C portion that are involved inthe interaction of the said fragment with an antigen-binding moleculethat binds to said (alpha)C portion. Such analysis is convenientlyperformed using recombinant DNA technology. In general, the DNA sequenceencoding the immuno-interactive fragment is cloned and manipulated sothat it may be expressed in a convenient host. DNA encoding theimmuno-interactive fragment can be obtained from a genomic library, fromcDNA derived from mRNA in cells expressing the said (alpha)C portion, orby synthetically constructing the DNA sequence (Sambrook et al., supra;Ausubel et al., supra).

[0141] The wild-type DNA encoding the immuno-interactive fragment isthen inserted into an appropriate plasmid or vector as described herein.In particular, prokaryotes are preferred for cloning and expressing DNAsequences to produce variants of the immuno-interactive fragment. Forexample, E. coli K12 strain 294 (ATCC No. 31446) may be used, as well asE. coli B, E. coli X1776 (ATCC No. 31537), and E. coli c600 and c60Ohfl,and E. coli W3110 (F⁻, gamma⁻, prototrophic, ATCC No. 27325), bacillisuch as Bacillus subtilis, and other enterobacteriaceae such asSalmonella typhimurium or Serratia marcescens, and various Pseudomonasspecies. A preferred prokaryote is E. coli W3110 (ATCC 27325).

[0142] Once the immuno-interactive fragment is cloned, site-specificmutagenesis as for example described by Carter et al. (1986, Nucl. AcidsRes., 13:4331) or by Zoller et al. (1987, Nucl. Acids Res., 10:6487),cassette mutagenesis as for example described by Wells et al. (1985,Gene 34:315), restriction selection mutagenesis as for example describedby Wells et al. (1986, Philos. Trans. R. Soc. London Ser. A 317:415), orother known techniques may be performed on the cloned DNA to produce thevariant DNA that encodes for the changes in amino acid sequence definedby the residues being substituted. When operably linked to anappropriate expression vector, variants are obtained. In some cases,recovery of the variant may be facilitated by expressing and secretingsuch molecules from the expression host by use of an appropriate signalsequence operably linked to the DNA sequence encoding theimmuno-interactive fragment parent or variant. Such methods are wellknown to those skilled in the art. Of course, other methods may beemployed to produce such polypeptides such as the in vitro chemicalsynthesis of the desired immuno-interactive fragment variant (Barany etal., 1979, In The Peptides, Gross et al., Eds., Academic Press, NewYork, Vol. 2, pp. 3-254).

[0143] Once the different the variants are produced, they are contactedwith an antigen-binding molecule that binds to the said (alpha)C portionand the interaction, if any, between the antigen-binding molecule andeach variant is determined. These activities are compared to theactivity of the wild-type immuno-interactive fragment with the sameantigen-binding molecule to determine which of the amino acid residuesin the active domain or epitope are involved in the interaction with theantigen-binding molecule. The scanning amino acid used in such ananalysis may be any different amino acid from that substituted, i.e.,any of the 19 other naturally occurring amino acids.

[0144] The interaction between the antigen-binding molecule and parentand variant can be measured by any convenient assay as for exampledescribed herein. While any number of analytical measurements may beused to compare activities, a convenient one for binding ofantigen-binding molecule is the dissociation constant K_(d) of thecomplex formed between the variant and antigen-binding molecule ascompared to the K_(d) for the wild-type immuno-interactive fragment.Generally, a two-fold increase or decrease in K_(d) per analogousresidue substituted by the substitution indicates that the substitutedresidue(s) is active in the interaction of the wild-typeimmuno-interactive fragment with the target antigen-binding molecule.

[0145] When a suspected or known active amino acid residue is subjectedto scanning amino acid analysis, the amino acid residues immediatelyadjacent thereto should be scanned. Three residue-substitutedpolypeptides can be made. One contains a scanning amino acid, preferablyalanine, at position N that is the suspected or known active amino acid.The two others contain the scanning amino acid at position N+1 and N−1.If each substituted immuno-interactive fragment causes a greater thanabout two-fold effect on K_(d) for the receptor, the scanning amino acidis substituted at position N+2 and N−2. This is repeated until at leastone, and preferably four, residues are identified in each directionwhich have less than about a two-fold effect on K_(d) or either of theends of the wild-type immuno-interactive fragment are reached. In thismanner, one or more amino acids along a continuous amino acid sequencethat are involved in the interaction with the particular antigen-bindingmolecule can be identified.

[0146] The active amino acid residue identified by amino acid scan istypically one that contacts the receptor target directly. However,active amino acids may also indirectly contact the target through saltbridges formed with other residues or small molecules such as H₂O orionic species such as Na⁺, Ca⁺², Mg⁺², or Zn⁺².

[0147] In some cases, the substitution of a scanning amino acid at oneor more residues results in a residue-substituted polypeptide which isnot expressed at levels that allow for the isolation of quantitiessufficient to carry out analysis of its activity with the receptor. Insuch cases, a different scanning amino acid, preferably an isostericamino acid, can be used.

[0148] Among the preferred scanning amino acids are relatively small,neutral amino acids. Such amino acids include alanine, glycine, serine,and cysteine. Alanine is the preferred scanning amino acid among thisgroup because it eliminates the side-chain beyond the beta-carbon and isless likely to alter the main-chain conformation of the variant. Alanineis also preferred because it is the most common amino acid. Further, itis frequently found in both buried and exposed positions (Creighton, TheProteins, W. H. Freeman & Co., New York.; Chothia, 1976, J. Mol. Biol.150:1). If alanine substitution does not yield adequate amounts ofvariant, an isosteric amino acid can be used. Alternatively, thefollowing amino acids in decreasing order of preference may be used:Ser, Asn, and Leu.

[0149] Once the active amino acid residues are identified, isostericamino acids may be substituted. Such isosteric substitutions need notoccur in all instances and may be performed before any active amino acidis identified. Such isosteric amino acid substitution is performed tominimize the potential disruptive effects on conformation that somesubstitutions can cause. Isosteric amino acids are shown in the tablebelow: TABLE B Polypeptide Amino Acid Isosteric Scanning Amino Acid Ala(A) Ser, Gly Glu (E) Gln, Asp Gln (Q) Asn, Glu Asp (D) Asn, Glu Asn (N)Ala, Asp Leu (L) Met, Ile Gly (G) Pro, Ala Lys (K) Met, Arg Ser (S) Thr,Ala Val (V) Ile, Thr Arg (R) Lys, Met, Asn Thr (T) Ser, Val Pro (P) GlyIle (I) Met, Leu, Val Met (M) Ile, Leu Phe (F) Tyr Tyr (Y) Phe Cys (C)Ser, Ala Trp (W) Phe His (H) Asn, Gln

[0150] The method herein can be used to detect active amino acidresidues within different epitopes of an immuno-interactive fragmentaccording to the invention. Once this identification is made, variousmodifications to the wild-type immuno-interactive fragment may be madeto modify the interaction between the parent immuno-interactive fragmentand one or more of the targets.

[0151] 2.3.2.4. Polypeptide or Peptide Libraries Produced by PhageDisplay

[0152] The identification of variants can also be facilitated throughthe use of a phage (or phagemid) display protein ligand screening systemas for example described by Lowman, et al. (1991, Biochemistry30:10832-10838), Markland, et al. (1991, Gene 109:13-19), Roberts, etal. (1992, Proc. Natl. Acad. Sci. USA 89:2429-2433), Smith (1985,Science 228:1315-1317), Smith et al. (1990, Science 248:1126-1128) andLardner et al. (U.S. Pat. No. 5,223,409). In general, this methodinvolves expressing a fusion protein in which the desired protein ligandis fused to the N-terminus of a viral coat protein (such as the M13 GeneIII coat protein, or a lambda coat protein).

[0153] In one embodiment, a library of phage is engineered to displaynovel peptides within the phage coat protein sequences. Novel peptidesequences are generated by random mutagenesis of gene fragments encodingan immuno-interactive polypeptide fragment using error-prone PCR, or byin vivo mutation by E. coli mutator cells. The novel peptides displayedon the surface of the phage are placed in contact, with an antigenbinding molecule such as an antibody or antibody fragment against theparticular immuno-interactive fragment on which the novel peptidesequences are based. Phage that display coat protein having peptidesthat are capable of binding to such antibodies are immobilized by suchtreatment, whereas all other phage can be washed away. After the removalof unbound phage, the bound phage can be amplified, and the DNA encodingtheir coat proteins can be sequenced. In this manner, the amino acidsequence of the embedded peptide or polypeptide can be deduced.

[0154] In more detail, the method involves:

[0155] (a) constructing a replicable expression vector comprising afirst polynucleotide encoding an immuno-interactive fragment of theinvention, a second polynucleotide encoding at least a portion of anatural or wild-type phage coat protein wherein the first and secondpolynucleotides are heterologous, and a transcription regulatory elementoperably linked to the first and second polynucleotides, thereby forminga polynucleotide fusion encoding a fusion protein;

[0156] (b) mutating the vector at one or more selected positions withinthe first polynucleotide thereby forming a family of related vectors;

[0157] (c) transforming suitable host cells with the vectors;

[0158] (d) infecting the transformed host cells with a helper phagehaving a polynucleotide encoding the phage coat protein;

[0159] (e) culturing the transformed infected host cells underconditions suitable for forming recombinant phagemid particlescontaining at least a portion of the vector and capable of transformingthe host, the conditions preferably adjusted so that no more than aminoramount of phagemid particles display more than one copy of the fusionprotein on the surface of the particle;

[0160] (f) contacting the phagemid particles with an antigen-bindingmolecule that binds to the immuno-interactive fragment so that at leasta portion of the phagemid particles bind to the antigen-bindingmolecule; and

[0161] (g) separating the phagemid particles that bind from those thatdo not.

[0162] Preferably, the method further comprises transforming suitablehost cells with recombinant phagemid particles that bind to theantigen-binding molecule and repeating steps (d) through (g) one or moretimes.

[0163] Preferably in this method the plasmid is under tight control ofthe transcription regulatory element, and the culturing conditions areadjusted so that the amount or number of phagemid particles displayingmore than one copy of the fusion protein on the surface of the particleis less than about 1%. Also, preferably, the amount of phagemidparticles displaying more than one copy of the fusion protein is lessthan 10% of the amount of phagemid particles displaying a single copy ofthe fusion protein. Even more preferably, the amount is less than 20%.

[0164] Typically in this method, the expression vector will furthercontain a secretory signal sequence fused to the DNA encoding eachsubunit of the polypeptide and the transcription regulatory element willbe a promoter. Preferred promoters are selected from lac Z, lambdaPL,tac, T7 polymerase, tryptophan, and alkaline phosphatase promoters andcombinations thereof. The method can also typically employ a helperphage selected from M13K07, M13R408, M13-VCS, and Phi X 174. Thepreferred helper phage is M13K07, and the preferred coat protein is theM13 Phage gene III coat protein. The preferred host is E. coli, andprotease-deficient strains of E. coli.

[0165] Repeated cycles of variant selection are used to select forhigher and higher affinity binding by the phagemid selection of multipleamino acid changes that are selected by multiple selection cycles.Following a first round of phagemid selection, involving a first regionor selection of amino acids in the ligand polypeptide, additional roundsof phagemid selection in other regions or amino acids of the ligandpolypeptide are conducted. The cycles of phagemid selection are repeateduntil the desired affinity properties of the ligand polypeptide areachieved.

[0166] It will be appreciated that the amino acid residues that form thebinding domain of the immuno-interactive fragment may not besequentially linked and may reside on different subunits of thepolypeptide. That is, the binding domain tracks with the particularsecondary structure at the binding site and not the primary structure.Thus, generally, mutations will be introduced into codons encoding aminoacids within a particular secondary structure at sites directed awayfrom the interior of the polypeptide so that they will have thepotential to interact with the antigen-binding molecule.

[0167] The phagemid-display method herein contemplates fusing apolynucleotide encoding the immuno-interactive fragment(polynucleotide 1) to a second polynucleotide (polynucleotide 2) suchthat a fusion protein is generated during transcription. Polynucleotide2 is typically a coat protein gene of a phage, and preferably it is thephage M13 gene III coat protein, or a fragment thereof. Fusion ofpolynucleotides 1 and 2 may be accomplished by inserting polynucleotide2 into a particular site on a vector that contains polynucleotide 1, orby inserting polynucleotide 1 into a particular site on a vector thatcontains polynucleotide 2.

[0168] Between polynucleotide 1 and polynucleotide 2, DNA encoding atermination codon may be inserted, which termination codons include UAG(amber), UAA (ocher), and UGA (opel) (see for example, Davis et al.,1980, Microbiology, Harper and Row, New York, pp. 237, 245-247, and274). The termination codon expressed in a wild-type host cell resultsin the synthesis of the polynucleotide 1 protein product without thepolynucleotide 2 protein fused thereto. However, growth in a suppressorhost cell results in the synthesis of detectable quantities of thefusion protein. In this regard, suppressor host cells contain a tRNAmodified to insert an amino acid in the termination codon position ofthe mRNA, thereby resulting in production of detectable amounts of thefusion protein. Such suppressor host cells are well known and described,such as E. coli suppressor strain (Bullock et al., 1987, BioTechniques,5:376-379). Any acceptable method may be used to place such atermination codon into the mRNA encoding the fusion polypeptide.

[0169] Accordingly, the suppressible codon can be inserted between thepolynucleotide encoding the immuno-interactive fragment and a secondpolynucleotide encoding at least a portion of a phage coat protein.Alternatively, the suppressible termination codon may be insertedadjacent to the fusion site by replacing the last amino acid triplet inthe polypeptide or the first amino acid triplet in the phage coatprotein. When the phagemid containing the suppressible codon is grown ina suppressor host cell, it results in the detectable production of afusion polypeptide containing the immuno-interactive fragment and thecoat protein. When the phagemid is grown in a non-suppressor host cell,the immuno-interactive fragment is synthesized substantially withoutfusion to the phage coat protein due to termination at the insertedsuppressible triplet encoding UAG, UAA, or UGA. In the non-suppressorcell the polypeptide is synthesized and secreted from the host cell dueto the absence of the fused phage coat protein which otherwise anchoredit to the host cell.

[0170] The immuno-interactive fragment may be altered at one or moreselected codons. An alteration is defined as a substitution, deletion,or insertion of one or more codons in the gene encoding theimmuno-interactive fragment that results in a change in the amino acidsequence of the immuno-interactive fragment as compared with theunaltered or native sequence of the said fragment. Preferably, thealterations will be by substitution of at least one amino acid with anyother amino acid in one or more regions of the molecule. The alterationsmay be produced by a variety of methods known in the art. These methodsinclude, but are not limited to, oligonucleotide-mediated mutagenesisand cassette mutagenesis as described for example herein.

[0171] For preparing the antigen-binding molecule and binding it withthe phagemid, the antigen-binding molecule is attached to a suitablematrix such as agarose beads, acrylamide beads, glass beads, cellulose,various acrylic copolymers, hydroxyalkyl methacrylate gels, polyacrylicacid, polymethacrylic copolymers, nylon, neutral and ionic carriers, andthe like. Attachment of the antigen-binding molecule to the matrix maybe accomplished by methods described in Methods Enzymol., 44:(1976), orby other means known in the art.

[0172] After attachment of the antigen-binding molecule to the matrix,the immobilized target is contacted with the library of phagemidparticles under conditions suitable for binding of at least a portion ofthe phagemid particles with the immobilized target. Normally, theconditions, including pH, ionic strength, temperature, and the like willmimic physiological conditions.

[0173] Bound phagemid particles (“binders”) having high affinity for theimmobilized target are separated from those having a low affinity (andthus do not bind to the target) by washing. Binders may be dissociatedfrom the immobilized target by a variety of methods. These methodsinclude competitive dissociation using the wild-type ligand, altering pHand/or ionic strength, and methods known in the art.

[0174] Suitable host cells are infected with the binders and helperphage, and the host cells are cultured under conditions suitable foramplification of the phagemid particles. The phagemid particles are thencollected and the selection process is repeated one or more times untilbinders having the desired affinity for the target molecule areselected.

[0175] 2.3.2.5. Rational Drug Design

[0176] Variants of naturally occurring immuno-interactive polypeptidesor polypeptide fragments according to the invention may also be obtainedusing the principles of conventional or of rational drug design as forexample described by Andrews, et al. (In: “Proceedings of the AlfredBenzon Symposium,” vol. 28, pp. 145-165, Munksgaard, Copenhagen, 1990),McPherson (1990, Eur. J. Biochem. 189:1-24), Hol et al. (1989, In:“Molecular Recognition: Chemical and Biochemical Problems”, Roberts,Ed., R. Soc. Chem. pp. 84-93), Hol (1989, Arzneim-Forsch. 39:1016-1018),Hol (1986, Angew. Chem. Int. Ed. Engl. 25:767-778).

[0177] In accordance with the methods of conventional drug design, thedesired variant molecules are obtained by randomly testing moleculeswhose structures have an attribute in common with the structure of a“native” immuno-interactive fragment according to the invention. Thequantitative contribution that results from a change in a particulargroup of a binding molecule can be determined by measuring the capacityof competition or cooperativity between the native immuno-interactivepolypeptide or polypeptide fragment and the putative polypeptidevariant.

[0178] In one embodiment of rational drug design, the polypeptidevariant is designed to share an attribute of the most stablethree-dimensional conformation of an immuno-interactive polypeptide orpolypeptide fragment according to the invention. Thus, the variant maybe designed to possess chemical groups that are oriented in a waysufficient to cause ionic, hydrophobic, or van der Waals interactionsthat are similar to those exhibited by the immuno-interactivepolypeptide or polypeptide fragment. In a second method of rationaldesign, the capacity of a particular immuno-interactive polypeptide orpolypeptide fragment to undergo conformational “breathing” is exploited.Such “breathing”—the transient and reversible assumption of a differentmolecular conformation—is a well-appreciated phenomenon, and resultsfrom temperature, thermodynamic factors, and from the catalytic activityof the molecule. Knowledge of the 3-dimensional structure of theimmuno-interactive polypeptide or polypeptide fragment facilitates suchan evaluation. An evaluation of the natural conformational changes of animmuno-interactive polypeptide or polypeptide fragment facilitates therecognition of potential hinge sites, potential sites at which hydrogenbonding, ionic bonds or van der Waals bonds might form or might beeliminated due to the breathing of the molecule, etc. Such recognitionpermits the identification of the additional conformations that theimmuno-interactive polypeptide or polypeptide fragment could assume, andenables the rational design and production of immunomimetics that sharesuch conformations.

[0179] The preferred method for performing rational immunomimetic designemploys a computer system capable of forming a representation of thethree-dimensional structure of the immuno-interactive polypeptide orpolypeptide fragment (such as those obtained using RIBBON (Priestle, J.,1988, J. Mol. Graphics 21:572), QUANTA (Polygen), InSite (Biosyn), orNanovision (American Chemical Society)). Such analyses are exemplifiedby Hol et al. (In: “Molecular Recognition: Chemical and BiochemicalProblems”, supra, Hol (1989, supra) and Hol (1986, supra).

[0180] In lieu of such direct comparative evaluations of putativepolypeptide variants, screening assays may be used to identify suchmolecules. Such assays will preferably exploit the capacity of thevariant to bind to an antigen-binding molecule as described in Section2.3.1.

[0181] 2.4. Polypeptide Derivatives

[0182] With reference to suitable derivatives of the invention, suchderivatives include amino acid deletions and/or additions to theimmuno-interactive fragment or variant of the invention, wherein saidderivatives elicit an immune response in a mammal which responseincludes elements that specifically bind to the said (alpha)C portion.“Additions” of amino acids may include fusion of the immuno-interactivefragments and polypeptide variants of the invention with otherpolypeptides or proteins. For example, it will be appreciated that saidimmuno-interactive fragments or variants may be incorporated into largerpolypeptides, and that such larger polypeptides may also be expected toelicit the said immune response.

[0183] The immuno-interactive fragments or variants of the invention maybe fused to a further protein, for example, which is not derived fromthe original host. The further protein may assist in the purification ofthe fusion protein. For instance, a polyhistidine tag or a maltosebinding protein may be used in this respect as described in more detailbelow. Other possible fusion proteins are those which produce animmunomodulatory response. Particular examples of such proteins includeProtein A or glutathione S-transferase (GST).

[0184] Other derivatives contemplated by the invention include, but arenot limited to, modification to side chains, incorporation of unnaturalamino acids and/or their derivatives during peptide, polypeptide orprotein synthesis and the use of crosslinkers and other methods whichimpose conformational constraints on the immuno-interactive fragmentsand variants of the invention.

[0185] Examples of side chain modifications contemplated by the presentinvention include modifications of amino groups such as by acylationwith acetic anhydride; acylation of amino groups with succinic anhydrideand tetrahydrophthalic anhydride; amidination with methylacetimidate;carbamoylation of amino groups with cyanate; pyridoxylation of lysinewith pyridoxal-5-phosphate followed by reduction with NaBH₄; reductivealkylation by reaction with an aldehyde followed by reduction withNaBH₄; and trinitrobenzylation of amino groups with2,4,6-trinitrobenzene sulphonic acid (TNBS).

[0186] The carboxyl group may be modified by carbodiimide activation viaO-acylisourea formation followed by subsequent derivitization, by way ofexample, to a corresponding amide.

[0187] The guanidine group of arginine residues may be modified byformation of heterocyclic condensation products with reagents such as2,3-butanedione, phenylglyoxal and glyoxal.

[0188] Sulfhydryl groups may be modified by methods such as performicacid oxidation to cysteic acid; formation of mercurial derivatives using4-chloromercuriphenylsulphonic acid, 4-chloromercuribenzoate;2-chloromercuri-4-nitrophenol, phenylmercury chloride, and othermercurials; formation of a mixed disulfides with other thiol compounds;reaction with maleimide, maleic anhydride or other substitutedmaleimide; carboxymethylation with iodoacetic acid or iodoacetamide; andcarbamoylation with cyanate at alkaline pH.

[0189] Tryptophan residues may be modified, for example, by alkylationof the indole ring with 2-hydroxy-5-nitrobenzyl bromide or sulfonylhalides or by oxidation with N-bromosuccinimide.

[0190] Tyrosine residues may be modified by nitration withtetranitromethane to form a 3-nitrotyrosine derivative.

[0191] The imidazole ring of a histidine residue may be modified byN-carbethoxylation with diethylpyrocarbonate or by alkylation withiodoacetic acid derivatives.

[0192] Examples of incorporating unnatural amino acids and derivativesduring peptide synthesis include but are not limited to, use of 4-aminobutyric acid, 6-aminohexanoic acid, 4-amino-3-hydroxy-5-phenylpentanoicacid, 4-amino-3-hydroxy-6-methylheptanoic acid, t-butylglycine,norleucine, norvaline, phenylglycine, omithine, sarcosine, 2-thienylalanine and/or D-isomers of amino acids. A list of unnatural amino acidscontemplated by the present invention is shown in Table C. TABLE CNon-conventional amino acid alpha-aminobutyric acidalpha-amino-alpha-methylbutyrate Aminocyclopropane-carboxylateAminoisobutyric acid Aminonorbornyl-carboxylate CyclohexylalanineCyclopentylalanine L-N-methylisoleucine D-alanine D-arginine D-asparticacid D-cysteine D-glutamate D-glutamic acid D-histidine D-isoleucineD-leucine D-lysine D-methionine D-ornithine D-phenylalanine D-prolineD-serine D-threonine D-tryptophan D-tyrosine D-valineD-alpha-methylalanine D-alpha-methylarginine D-alpha-methylasparagineD-alpha-methylaspartate D-alpha-methylcysteine D-alpha-methylglutamineD-alpha-methylhistidine D-alpha-methylisoleucine D-alpha-methylleucineD-alpha-methyllysine D-alpha-methylmethionine D-alpha-methylornithiineD-alpha-methylphenylalanine D-alpha-methylproline D-alpha-methylserineD-alpha-methylthreonine D-alpha-methyltryptophan D-alpha-methyltyrosineL-alpha-methylleucine L-alpha-methylmethionine L-alpha-methylnorvatineL-alpha-methylphenylalanine L-alpha-methylserineL-alpha-methyltryptophan L-alpha-methylvaline N-(N-(2,2-diphenylethylcarbamylmethyl)glycine 1-carboxy-1-(2,2-diphenyl-ethylamino)cyclopropane L-N-methylalanine L-N-methylarginineL-N-methylasparagine L-N-methylaspartic acid L-N-methylcysteineL-N-methylglutamine L-N-methylglutamic acid L-N-methylhistidineL-N-methylleucine L-N-methyllysine L-N-methylmethionineL-N-methylnorleucine L-N-methylnorvaline L-N-methylornithineL-N-methylphenylalanine L-N-methylproline L-N-medlylserineL-N-methylthreonine L-N-methyltryptophan L-N-methyltyrosineL-N-methylvaline L-N-methylethylglycine L-N-methyl-t-butylglycineL-norleucine L-norvaline alpha-methyl-aminoisobutyratealpha-methyl-gamma-aminobutyrate alpha-methylcyclohexylalaninealpha-methylcylcopentylalanine alpha-methyl-alpha-napthylalaninealpha-methylpenicillamine N-(4-aminobutyl)glycineN-(2-aminoethyl)glycine N-(3-aminopropyl)glycineN-amino-alpha-methylbutyrate alpha-napthylalanine N-benzylglycineN-(2-carbamylediyl)glycine N-(carbamylmethyl)glycineN-(2-carboxyethyl)glycine N-(carboxymethyl)glycine N-cyclobutylglycineN-cycloheptylglycine N-cyclohexylglycine N-cyclodecylglycineL-alpha-methyllysine L-alpha-methylnorleucine L-alpha-methylornithineL-alpha-methylproline L-alpha-methylthreonine L-alpha-methyltyrosineL-N-methylhomophenylalanine N-(N-(3,3-diphenylpropylcarbamylmethyl)glycine

[0193] The invention also extends to covalently modifying animmuno-interactive fragment or variant of the invention withdinitrophenol, in order to render it immunogenic in humans.

[0194] Also contemplated is the use of crosslinkers, for example, tostabilize 3D conformations of the immuno-interactive fragments orvariants of the invention, using homo-bifunctional cross linkers such asbifunctional imido esters having (CH₂)_(n) spacer groups with n=1 ton=6, glutaraldehyde, N-hydroxysuccinimide esters and hetero-bifunctionalreagents which usually contain an amino-reactive moiety such asN-hydroxysuccinimide and another group specific-reactive moiety such asmaleimido or dithio moiety or carbodiimide. In addition, peptides can beconformationally constrained, for example, by introduction of doublebonds between C_(alpha) and C_(beta) atoms of amino acids, byincorporation of C_(alpha) and N_(alpha)-methylamino acids, and byformation of cyclic peptides or analogues by introducing covalent bondssuch as forming an amide bond between the N and C termini between twoside chains or between a side chain and the N or C terminus of thepeptides or analogues. For example, reference may be made to Marlowe(1993, Biorg. Med. Chem. Lett. 3:437-44) who describes peptidecyclization on TFA resin using trimethylsilyl (TMSE) ester as anorthogonal protecting group; Pallin and Tam (1995, J. Chem. Soc. Chem.Comm. 2021-2022) who describe the cyclization of unprotected peptides inaqueous solution by oxime formation; Algin et al (1994, TetrahedronLett. 35:9633-9636) who disclose solid-phase synthesis of head-to-tailcyclic peptides via lysine side-chain anchoring; Kates et al (1993,Tetrahedron Lett. 34:1549-1552) who describe the production ofhead-to-tail cyclic peptides by three-dimensional solid phase strategy;Tumelty et al (1994, J. Chem. Soc. Chem. Comm. 1067-1068) who describethe synthesis of cyclic peptides from an immobilized activatedintermediate, wherein activation of the immobilized peptide is carriedout with N-protecting group intact and subsequent removal leading tocyclization; McMurray et al (1994, Peptide Res. 7:195-206) who disclosehead-to-tail cyclization of peptides attached to insoluble supports bymeans of the side chains of aspartic and glutamic acid; Hruby et al(1994, Reactive Polymers 22:231-241) who teach an alternate method forcyclizing peptides via solid supports; and Schmidt and Langer (1997, J.Peptide Res. 49:67-73) who disclose a method for synthesizingcyclotetrapeptides and cyclopentapeptides. The foregoing methods may beused to produce conformationally constrained polypeptides that elicit animmune response against the said (alpha)C portion.

[0195] The invention also contemplates immuno-interactive fragments orvariants of the invention that have been modified using ordinarymolecular biological techniques so as to improve their resistance toproteolytic degradation or to optimize solubility properties or torender them more suitable as an immunogenic agent.

[0196] 2.5. Methods of Preparing the Polypeptides of the Invention

[0197] Polypeptides of the inventions may be prepared by any suitableprocedure known to those of skill in the art. For example, thepolypeptides may be prepared by a procedure including the steps of:

[0198] (a) preparing a recombinant polynucleotide comprising anucleotide sequence encoding an immuno-interactive fragment of thepolypeptide set forth in SEQ ID NO: 2 or preferably the polypeptide setforth in any one or more of SEQ ID NO: 3, 4, 5, 6, 18, 19, 20, 21, 22,23, 30, 31, 32, 35, 36, 37, 38, 39, 40, 55, 56, 57, 58, 59, 60, 68, 69,70, 71, 72 and 73, or a variant or derivative of these, which nucleotidesequence is operably linked to a regulatory polynucleotide whichtypically comprises transcriptional and translational regulatory nucleicacid;

[0199] (b) introducing the recombinant polynucleotide into a suitablehost cell;

[0200] (c) culturing the host cell to express recombinant polypeptidefrom said recombinant polynucleotide; and

[0201] (d) isolating the recombinant polypeptide.

[0202] The recombinant polynucleotide preferably comprises either anexpression vector that may be a self-replicating extra-chromosomalvector such as a plasmid, or a vector that integrates into a hostgenome.

[0203] The transcriptional and translational regulatory nucleic acidwill generally be appropriate for the host cell used for expression.Numerous types of appropriate expression vectors and suitable regulatorysequences are known in the art for a variety of host cells.

[0204] Typically, the transcriptional and translational regulatorynucleic acid may include, but is not limited to, promoter sequences,leader or signal sequences, ribosomal binding sites, transcriptionalstart and stop sequences, translational start and termination sequences,and enhancer or activator sequences.

[0205] Constitutive or inducible promoters as known in the art arecontemplated by the invention. The promoters may be either naturallyoccurring promoters, or hybrid promoters that combine elements of morethan one promoter.

[0206] In a preferred embodiment, the expression vector contains aselectable marker gene to allow the selection of transformed host cells.Selection genes are well known in the art and will vary with the hostcell used.

[0207] The expression vector may also include a fusion partner(typically provided by the expression vector) so that the recombinantpolypeptide of the invention is expressed as a fusion polypeptide withsaid fusion partner. The main advantage of fusion partners is that theyassist identification and/or purification of said fusion polypeptide.

[0208] In order to express said fusion polypeptide, it is necessary toligate a polynucleotide according to the invention into the expressionvector so that the translational reading frames of the fusion partnerand the polynucleotide coincide.

[0209] Well known examples of fusion partners include, but are notlimited to, glutathione-S-transferase (GST), Fc potion of human IgG,maltose binding protein (MBP) and hexahistidine (HIS6), which areparticularly useful for isolation of the fusion polypeptide by affinitychromatography. For the purposes of fusion polypeptide purification byaffinity chromatography, relevant matrices for affinity chromatographyare glutathione-, amylose-, and nickel- or cobalt-conjugated resinsrespectively. Many such matrices are available in “kit” form, such asthe QIAexpress™ system (Qiagen) useful with (HIS6) fusion partners andthe Pharmacia GST purification system.

[0210] Another fusion partner well known in the art is green fluorescentprotein (GFP). This fusion partner serves as a fluorescent “tag” whichallows the fusion polypeptide of the invention to be identified byfluorescence microscopy or by flow cytometry. The GFP tag is useful whenassessing sub-cellular localization of the fusion polypeptide of theinvention, or for isolating cells which express the fusion polypeptideof the invention. Flow cytometric methods such as fluorescence activatedcell sorting (FACS) are particularly useful in this latter application.

[0211] Preferably, the fusion partners also have protease cleavagesites, such as for Factor X_(a) or Thrombin, which allow the relevantprotease to partially digest the fusion polypeptide of the invention andthereby liberate the recombinant polypeptide of the invention therefrom.The liberated polypeptide can then be isolated from the fusion partnerby subsequent chromatographic separation.

[0212] Fusion partners according to the invention also include withintheir scope “epitope tags”, which are usually short peptide sequencesfor which a specific antibody is available. Well known examples ofepitope tags for which specific monoclonal antibodies are readilyavailable include c-Myc, influenza virus, hemagglutinin and FLAG™ tags.

[0213] The step of introducing into the host cell the recombinantpolynucleotide may be effected by any suitable method includingtransfection, and transformation, the choice of which will be dependenton the host cell employed. Such methods are well known to those of skillin the art.

[0214] Recombinant polypeptides of the invention may be produced byculturing a host cell transformed with an expression vector containingnucleic acid encoding an immuno-interactive fragment, variant orderivative according to the invention. The conditions appropriate forprotein expression will vary with the choice of expression vector andthe host cell. This is easily ascertained by one skilled in the artthrough routine experimentation.

[0215] Suitable host cells for expression may be prokaryotic oreukaryotic. One preferred host cell for expression of a polypeptideaccording to the invention is a bacterium. The bacterium used may beEscherichia coli. Alternatively, the host cell may be an insect cellsuch as, for example, SF9 cells that may be utilized with a baculovirusexpression system.

[0216] The recombinant protein may be conveniently prepared by a personskilled in the art using standard protocols as for example described inSambrook, et al., Molecular Cloning, a Laboratory Manual (Cold SpringHarbor Press, 1989), in particular Sections 16 and 17; Ausubel et al.,Current Protocols in Molecular Biology (John Wiley & Sons, Inc.1994-1998), in particular Chapters 10 and 16; and Coligan et al.,Current Protocols in Protein Science (John Wiley & Sons, Inc.1995-1997), in particular Chapters 1, 5 and 6.

[0217] In some cases, the recombinant polypeptide may require refolding.Methods of refolding are well known to those of skill in the art.

[0218] Alternatively, the polypeptide fragments, variants or derivativesof the invention may be synthesized using solution synthesis or solidphase synthesis as described, for example, in Chapter 9 of Atherton andShephard (supra).

[0219] 3. Polynucleotides of the Invention

[0220] 3.1. Polynucleotides Encoding Immuno-Interactive Fragments of theInvention

[0221] The invention further provides a polynucleotide that encodes animmuno-interactive fragment, variant or derivative as defined above.Suitably, the polynucleotide comprises a fragment of the full-lengthnucleic acid sequence encoding the (alpha)C portion of the human inhibinalpha-subunit which fragment encodes an immuno-interactive fragmentaccording to the invention. In this regard, reference may be made to SEQID NO: 1, which corresponds to nucleotide 841 through nucleotide 1245 ofthe full-length human inhibin alpha-subunit mRNA as for exampledisclosed under Accession No. M13981 of the GenBank database (supra),and which encodes the (alpha)C portion of the human inhibinalpha-subunit.

[0222] Preferably, the polynucleotide comprises a nucleic acid sequenceencoding a polypeptide according to any one or more of SEQ ID NOs: 3, 4,5, 6, 18, 19, 20, 21, 22, 23, 30, 31, 32, 35, 36, 37, 38, 39, 40, 55,56, 57, 58, 59, 60, 68, 69, 70, 71, 72 and 73. Conveniently, suchpolynucleotide can be obtained from SEQ ID NO: 1, which encodes the(alpha)C portion of the human inhibin alpha-subunit set forth in SEQ IDNO: 2.

[0223] 3.2. Polynucleotides Variants

[0224] In general, polynucleotide variants according to the inventioncomprise regions that show at least 60%, more suitably at least 70%,preferably at least 80%, and most preferably at least 90% sequenceidentity over a reference polynucleotide sequence of identical size(“comparison window”) or when compared to an aligned sequence in whichthe alignment is performed by a computer homology program known in theart. What constitutes suitable variants may be determined byconventional techniques. For example, a polynucleotide fragment of SEQID NO: 1 or a polynucleotide encoding a polypeptide according to any oneor more of SEQ ID NOs: 3, 4, 5, 6, 18, 19, 20, 21, 22, 23, 30, 31, 32,35, 36, 37, 38, 39, 40, 55, 56, 57, 58, 59, 60, 68, 69, 70, 71, 72 and73 can be mutated using random mutagenesis (e.g., transposonmutagenesis), oligonucleotide-mediated (or site-directed) mutagenesis,PCR mutagenesis and cassette mutagenesis of an earlier prepared variantor non-variant version of an isolated natural promoter according to theinvention.

[0225] Oligonucleotide-mediated mutagenesis is a preferred method forpreparing nucleotide substitution variants of a polynucleotide of theinvention. This technique is well known in the art as, for example,described by Adelman et al. (1983, DNA 2:183). Briefly, a polynucleotidefragment of SEQ ID NO: 1 or a polynucleotide encoding a polypeptideaccording to any one or more of SEQ ID NOs: 3, 4, 5, 6, 18, 19, 20, 21,22, 23, 30, 31, 32, 35, 36, 37, 38, 39, 40, 55, 56, 57, 58, 59, 60, 68,69, 70, 71, 72 and 73 is altered by hybridizing an oligonucleotideencoding the desired mutation to a template DNA, where the template isthe single-stranded form of a plasmid or bacteriophage containing theunaltered or parent DNA sequence. After hybridization, a DNA polymeraseis used to synthesize an entire second complementary strand of thetemplate that will thus incorporate the oligonucleotide primer, and willcode for the selected alteration in said parent DNA sequence.

[0226] Generally, oligonucleotides of at least 25 nucleotides in lengthare used. An optimal oligonucleotide will have 12 to 15 nucleotides thatare completely complementary to the template on either side of thenucleotide(s) coding for the mutation. This ensures that theoligonucleotide will hybridize properly to the single-stranded DNAtemplate molecule.

[0227] The DNA template can be generated by those vectors that areeither derived from bacteriophage M13 vectors, or those vectors thatcontain a single-stranded phage origin of replication as described byViera et al. (1987, Methods Enzymol. 153:3). Thus, the DNA that is to bemutated may be inserted into one of the vectors to generatesingle-stranded template. Production of single-stranded template isdescribed, for example, in Sections 4.21-4.41 of Sambrook et al. (1989,supra).

[0228] Alternatively, the single-stranded template may be generated bydenaturing double-stranded plasmid (or other DNA) using standardtechniques.

[0229] For alteration of the native DNA sequence, the oligonucleotide ishybridized to the single-stranded template under suitable hybridizationconditions. A DNA polymerizing enzyme, usually the Klenow fragment ofDNA polymerase I, is then added to synthesize the complementary strandof the template using the oligonucleotide as a primer for synthesis. Aheteroduplex molecule is thus formed such that one strand of DNA encodesthe mutated form of the immuno-interactive fragment under test, and theother strand (the original template) encodes the native unalteredsequence of the immuno-interactive fragment under test. Thisheteroduplex molecule is then transformed into a suitable host cell,usually a prokaryote such as E. coli. After the cells are grown, theyare plated onto agarose plates and screened using the oligonucleotideprimer having a detectable label to identify the bacterial colonieshaving the mutated DNA. The resultant mutated DNA fragments are thencloned into suitable expression hosts such as E. coli using conventionaltechnology and clones that retain the desired antigenic activity aredetected. Where the clones have been derived using random mutagenesistechniques, positive clones would have to be sequenced in order todetect the mutation.

[0230] Alternatively, linker-scanning mutagenesis of DNA may be used tointroduce clusters of point mutations throughout a sequence of interestthat has been cloned into a plasmid vector. For example, reference maybe made to Ausubel et al., supra, (in particular, Chapter 8.4) whichdescribes a first protocol that uses complementary oligonucleotides andrequires a unique restriction site adjacent to the region that is to bemutagenized. A nested series of deletion mutations is first generated inthe region. A pair of complementary oligonucleotides is synthesized tofill in the gap in the sequence of interest between the linker at thedeletion endpoint and the nearby restriction site. The linker sequenceactually provides the desired clusters of point mutations as it is movedor “scanned” across the region by its position at the varied endpointsof the deletion mutation series. An alternate protocol is also describedby Ausubel et al., supra, which makes use of site directed mutagenesisprocedures to introduce small clusters of point mutations throughout thetarget region. Briefly, mutations are introduced into a sequence byannealing a synthetic oligonucleotide containing one or more mismatchesto the sequence of interest cloned into a single-stranded M13 vector.This template is grown in an E. coli dut⁻ ung⁻ strain, which allows theincorporation of uracil into the template strand. The oligonucleotide isannealed to the template and extended with T4 DNA polymerase to create adouble-stranded heteroduplex. Finally, the heteroduplex is introducedinto a wild-type E. coli strain, which will prevent replication of thetemplate strand due to the presence of apurinic sites (generated whereuracil is incorporated), thereby resulting in plaques containing onlymutated DNA.

[0231] Region-specific mutagenesis and directed mutagenesis using PCRmay also be employed to construct polynucleotide variants according tothe invention. In this regard, reference may be made, for example, toAusubel et al., supra, in particular Chapters 8.2A and 8.5.

[0232] Alternatively, suitable polynucleotide sequence variants of theinvention may be prepared according to the following procedure:

[0233] (a) creating primers which are optionally degenerate wherein eachcomprises a portion of a reference polynucleotide encoding a referenceimmuno-interactive fragment of the invention, preferably encoding thesequence set forth in any one or more of SEQ ID NO: 3, 4, 5, 6, 18, 19,20, 21, 22, 23, 30, 31, 32, 35, 36, 37, 38, 39, 40, 55, 56, 57, 58, 59,60, 68, 69, 70, 71, 72 and 73;

[0234] (b) obtaining a nucleic acid extract from a different mammal fromwhich said reference polynucleotide is derived; and

[0235] (c) using said primers to amplify, via nucleic acid amplificationtechniques, at least one amplification product from said nucleic acidextract, wherein said amplification product corresponds to apolynucleotide variant.

[0236] Suitable nucleic acid amplification techniques are well known tothe skilled addressee, and include polymerase chain reaction (PCR) asfor example described in Ausubel et al. (supra); strand displacementamplification (SDA) as for example described in U.S. Pat. No. 5,422,252;rolling circle replication (RCR) as for example described in Liu et al.,(1996, J. Am. Chem. Soc. 118:1587-1594 and International application WO92/01813) and Lizardi et al., (international Application WO 97/19193);nucleic acid sequence-based amplification (NASBA) as for exampledescribed by Sooknanan et al., (1994, Biotechniques 17:1077-1080); andQ-beta replicase amplification as for example described by Tyagi et al.,(1996, Proc. Natl. Acad. Sci. USA 93:5395-5400).

[0237] Typically, polynucleotide variants that are substantiallycomplementary to a reference polynucleotide are identified by blottingtechniques that include a step whereby nucleic acids are immobilized ona matrix (preferably a synthetic membrane such as nitrocellulose),followed by a hybridization step, and a detection step. Southernblotting is used to identify a complementary DNA sequence; northernblotting is used to identify a complementary RNA sequence. Dot blottingand slot blotting can be used to identify complementary DNA/DNA, DNA/RNAor RNA/RNA polynucleotide sequences. Such techniques are well known bythose skilled in the art, and have been described in Ausubel et al.(1994-1998, supra) at pages 2.9.1 through 2.9.20.

[0238] According to such methods, Southern blotting involves separatingDNA molecules according to size by gel electrophoresis, transferring thesize-separated DNA to a synthetic membrane, and hybridizing themembrane-bound DNA to a complementary nucleotide sequence labeledradioactively, enzymatically or fluorochromatically. In dot blotting andslot blotting, DNA samples are directly applied to a synthetic membraneprior to hybridization as above.

[0239] An alternative blotting step is used when identifyingcomplementary polynucleotides in a cDNA or genomic DNA library, such asthrough the process of plaque or colony hybridization. A typical exampleof this procedure is described in Sambrook et al. (“Molecular Cloning, ALaboratory Manual”, Cold Spring Harbour Press, 1989) Chapters 8-12.

[0240] Typically, the following general procedure can be used todetermine hybridization conditions. Polynucleotides areblotted/transferred to a synthetic membrane, as described above. Areference polynucleotide such as a polynucleotide of the invention islabeled as described above, and the ability of this labeledpolynucleotide to hybridize with an immobilized polynucleotide isanalyzed.

[0241] A skilled addressee will recognize that a number of factorsinfluence hybridization. The specific activity of radioactively labeledpolynucleotide sequence should typically be greater than or equal toabout 108 dpm/mg to provide a detectable signal. A radiolabelednucleotide sequence of specific activity 108 to 109 dpm/mg can detectapproximately 0.5 pg of DNA. It is well known in the art that sufficientDNA must be immobilized on the membrane to permit detection. It isdesirable to have excess immobilized DNA, usually 10 micrograms. Addingan inert polymer such as 10% (w/v) dextran sulfate (MW 500,000) orpolyethylene glycol 6000 during hybridization can also increase thesensitivity of hybridization (see Ausubel supra at 2.10.10).

[0242] To achieve meaningful results from hybridization between apolynucleotide immobilized on a membrane and a labeled polynucleotide, asufficient amount of the labeled polynucleotide must be hybridized tothe immobilized polynucleotide following washing. Washing ensures thatthe labeled polynucleotide is hybridized only to the immobilizedpolynucleotide with a desired degree of complementarity to the labeledpolynucleotide.

[0243] It will be understood that polynucleotide variants according tothe invention will hybridize to a reference polynucleotide under atleast low stringency conditions. Reference herein to low stringencyconditions include and encompass from at least about 1% v/v to at leastabout 15% v/v formamide and from at least about 1 M to at least about 2M salt for hybridization at 42° C., and at least about 1 M to at leastabout 2 M salt for washing at 42° C. Low stringency conditions also mayinclude 1% Bovine Serum Albumin (BSA), 1 mM EDTA, 0.5 M NAHPO₄ (pH 7.2),7% SDS for hybridization at 65° C., and (i) 2×SSC, 0.1% SDS; or (ii)0.5% BSA, 1 mM EDTA, 40 mM NaHPO₄ (pH 7.2), 5% SDS for washing at roomtemperature.

[0244] Suitably, the polynucleotide variants hybridize to a referencepolynucleotide under at least medium stringency conditions. Mediumstringency conditions include and encompass from at least about 16% v/vto at least about 30% v/v formamide and from at least about 0.5 M to atleast about 0.9 M salt for hybridization at 42° C., and at least about0.5 M to at least about 0.9 M salt for washing at 42° C. Mediumstringency conditions also may include 1% Bovine Serum Albumin (BSA), 1mM EDTA, 0.5 M NAHPO₄ (pH 7.2), 7% SDS for hybridization at 65° C., and(i) 2×SSC, 0.1% SDS; or (ii) 0.5% BSA, 1 mM EDTA, 40 mM NaHPO₄ (pH 7.2),5% SDS for washing at 42° C.

[0245] Preferably, the polynucleotide variants hybridize to a referencepolynucleotide under high stringency conditions. High stringencyconditions include and encompass from at least about 31% v/v to at leastabout 50% v/v formamide and from at least about 0.01 M to at least about0.15 M salt for hybridization at 42° C., and at least about 0.01 M to atleast about 0.15 M salt for washing at 42° C. High stringency conditionsalso may include 1% BSA, 1 MM EDTA, 0.5 M NaHPO₄ (pH 7.2), 7% SDS forhybridization at 65° C., and (i) 0.2×SSC, 0.1% SDS; or (ii) 0.5% BSA, 1mM EDTA, 40 mM NaHPO₄ (pH 7.2), 1% SDS for washing at a temperature inexcess of 65° C.

[0246] Other stringent conditions are well known in the art. A skilledaddressee will recognize that various factors can be manipulated tooptimize the specificity of the hybridization. Optimization of thestringency of the final washes can serve to ensure a high degree ofhybridization. For detailed examples, see Ausubel et al., supra at pages2.10.1 to 2.10.16 and Sambrook et al. (1989, supra) at sections 1.101 to1.104.

[0247] While stringent washes are typically carried out at temperaturesfrom about 42° C. to 68° C., one skilled in the art will appreciate thatother temperatures may be suitable for stringent conditions. Maximumhybridization typically occurs at about 20° C. to 25° C. below the T_(m)for formation of a DNA-DNA hybrid. It is well known in the art that theT_(m) is the melting temperature, or temperature at which twocomplementary polynucleotide sequences dissociate. Methods forestimating T_(m) are well known in the art (see Ausubel et al., supra atpage 2.10.8).

[0248] In general, washing is carried out at T=69.3+0.41 (G+C) % −12° C.However, the T_(m) of a duplex DNA decreases by 1° C. with everyincrease of 1% in the number of mismatched base pairs.

[0249] In a preferred hybridization procedure, a membrane (e.g., anitrocellulose membrane or a nylon membrane) containing immobilized DNAis hybridized overnight at 42° C. in a hybridization buffer (50%deionized formamide, 5×SSC, 5× Denhardt's solution (0.1% Ficoll, 0.1%polyvinylpyrollidone and 0.1% bovine serum albumin), 0.1% SDS and 200mg/mL denatured salmon sperm DNA) containing labeled probe. The membraneis then subjected to two sequential medium stringency washes (i.e.,2×SSC/0.1% SDS for 15 min at 45° C., followed by 2×SSC/0.1% SDS for 15min at 50° C.), followed by two sequential high stringency washes (i.e.,0.2×SSC/0.1% SDS for 12 min at 55° C. followed by 0.2×SSC and 0.1% SDSsolution for 12 min).

[0250] Methods for detecting a labeled polynucleotide hybridized to animmobilized polynucleotide are well known to practitioners in the art.Such methods include autoradiography, phosphorimaging, andchemiluminescent, fluorescent and calorimetric detection.

[0251] 4. Antigen-Binding Molecules

[0252] The invention also contemplates antigen-binding molecules againstthe aforementioned fragments, variants and derivatives. Antigen-bindingmolecules contemplated by the present invention include monoclonalantibodies. Such antibodies may be produced using the standard method asdescribed, for example, by Kohler and Milstein (1975, Nature 256,495-497), or by more recent modifications thereof as described, forexample, in Coligan et al., (1991, supra) by immortalizing spleen orother antibody producing cells derived from a production species whichhas been inoculated with one or more of the immuno-interactivefragments, variants or derivatives of the invention. Exemplary methodsfor producing monoclonal antibodies, which are immuno-interactive withthe polypeptides of the invention, are described in Groome et al. (1994,In “Inhibin and inhibin-related proteins,” Burger, Ed. Frontiers inEndocrinology, Vol. 3 Ares Serono Symposia) and in Groome et al. (1994,Clin. Endocrinol. 40:717-723).

[0253] The invention also contemplates as antigen-binding molecules Fv,Fab, Fab′ and F(ab′)₂ immunoglobulin fragments.

[0254] Alternatively, the antigen-binding molecule may comprise asynthetic stabilized Fv fragment. Exemplary fragments of this typeinclude single chain Fv fragments (sFv, frequently termed scFv) in whicha peptide linker is used to bridge the N terminus or C terminus of aV_(H) domain with the C terminus or N-terminus, respectively, of a V_(L)domain. ScFv lack all constant parts of whole antibodies and are notable to activate complement. Suitable peptide linkers for joining theV_(H) and V_(L) domains are those which allow the V_(H) and V_(L)domains to fold into a single polypeptide chain having an antigenbinding site with a three dimensional structure similar to that of theantigen binding site of a whole antibody from which the Fv fragment isderived. Linkers having the desired properties may be obtained by themethod disclosed in U.S. Pat. No. 4,946,778. However, in some cases alinker is absent. ScFvs may be prepared, for example, in accordance withmethods outlined in Krebber et al. (1997, J. Immunol. Meth.201(1):35-55). Alternatively, they may be prepared by methods describedin U.S. Pat. No. 5,091,513, European Patent No. 239,400 or the articlesby Winter and Milstein (1991, Nature 349:293) and Plückthun et al (1996,In Antibody engineering: A practical approach, pp. 203-252).

[0255] Alternatively, the synthetic stabilized Fv fragment comprises adisulfide stabilized Fv (dsFv) in which cysteine residues are introducedinto the V_(H) and V_(L) domains such that in the fully folded Fvmolecule the two residues will form a disulfide bond therebetween.Suitable methods of producing dsFv are described for example in(Glockscuther et al., Biochemistry 29:1363-1367; Reiter et al., 1994, J.Biol. Chem. 269:18327-18331; Reiter et al., 1994, Biochemistry33:5451-5459; Reiter et al., 1994, Cancer Res. 54:2714-2718; Webber etal., 1995, Mol. Immunol. 32:249-258).

[0256] Also contemplated as antigen-binding molecules are singlevariable region domains (termed dAbs) as for example disclosed in (Wardet al., 1989, Nature 341:544-546; Hamers-Casterman et al., 1993, Nature363:446-448; Davies et al., 1994, FEBS Lett. 339:285-290).

[0257] Alternatively, the antigen-binding molecule may comprise a“minibody.” In this regard, minibodies are small versions of wholeantibodies, which encode in a single chain the essential elements of awhole antibody. Suitably, the minibody is comprised of the V_(H) andV_(L) domains of a native antibody fused to the hinge region and CH3domain of the immunoglobulin molecule as, for example, disclosed in U.S.Pat. No. 5,837,821.

[0258] In an alternate embodiment, the antigen binding molecule maycomprise non-immunoglobulin derived, protein frameworks. For example,reference may be made to (Ku et al., 1995, Proc. Natl. Acad. Sci. USA92:652-6556) which discloses a four-helix bundle protein cytochrome b562having two loops randomized to create complementarity determiningregions (CDRs), which have been selected for antigen binding.

[0259] The antigen-binding molecule may be multivalent (i.e., havingmore than one antigen binding site). Such multivalent molecules may bespecific for one or more antigens. Multivalent molecules of this typemay be prepared by dimerization of two antibody fragments through acysteinyl-containing peptide as, for example disclosed by (Adams et al.,1993, Cancer Res. 53:4026-4034; Cumber et al., 1992, J. Immunol.149:120-126). Alternatively, dimerization may be facilitated by fusionof the antibody fragments to amphiphilic helices that naturally dimerize(Plunckthun, 1992, Biochemistry 31:1579-1584), or by use of domains(such as leucine zippers jun and fos) that preferentially heterodimerize(Kostelny et al., 1992, J. Immunol. 148:1547-1553).

[0260] In an alternate embodiment, the multivalent molecule may comprisea multivalent single chain antibody (multi-scFv) comprising at least twoscFvs linked together by a peptide linker. In this regard,non-covalently or covalently linked scFv dimers termed “diabodies” maybe used. Multi-scFvs may be bispecific or greater depending on thenumber of scFvs employed having different antigen-binding specificities.Multi-scFvs may be prepared for example by methods disclosed in U.S.Pat. No. 5,892,020.

[0261] The monoclonal antibodies, immunoglobulin fragments andimmunoglobulin-like fragments described above are particularly preferredas antigen-binding molecules to replace polyclonal antibodies used incurrent two-site assays for inhibin A, B and Pro-(alpha)C, as well asinhibin alpha subunit assays.

[0262] The antigen-binding molecules of the invention may be used foraffinity chromatography in isolating a natural or recombinant mammalianinhibin and in particular, a natural or recombinant mammalian inhibinalpha subunit. For example reference may be made to immunoaffinitychromatographic procedures described in Chapter 9.5 of Coligan et al.,(Current Protocols in Immunology, (John Wiley & Sons, Inc, 1991-1997).

[0263] The antigen-binding molecules can be used to screen expressionlibraries for variant polypeptides of the invention as described herein.They can also be used to detect mammalian inhibin, preferably mammalianinhibin alpha subunit, as described hereinafter. In addition, theantigen-binding molecules of the invention can be used to treat acondition associated with aberrant concentrations of the (alpha)Cportion of a mammalian inhibin alpha subunit in a biological sample, asdescribed hereinafter.

[0264] 5. Detection of Mammalian Inhibin

[0265] The presence or absence of a mammalian inhibin in a patient maybe determined by isolating the biological sample from the patient,contacting the biological sample with an antigen-binding molecule asdescribed in Section 4, and detecting the presence of a complexcomprising the said antigen-binding molecule and the mammalian inhibin.In this regard, the antigen-binding molecule may be species-specific,that is specific to an inhibin of a particular mammal. Preferably, theantigen-binding molecule detects inhibin from a plurality of mammalianspecies.

[0266] There is also provided a method of diagnosing a conditionassociated with an aberrant concentration of a mammalian inhibin in abiological sample of a patient. The method comprises contacting thebiological sample with an antigen-binding molecule as described inSection 4, measuring the concentration of a complex comprising the saidantigen-binding molecule and the mammalian inhibin in said contactedsample, and relating said measured complex concentration to theconcentration of mammalian inhibin in said sample, wherein the presenceof said aberrant concentration is indicative of said condition.Suitably, the condition is a cancer, more preferably anendocrine-related cancer. Preferably, the endocrine-related cancer is acancer of a reproductive organ. In a preferred embodiment, theendocrine-related cancer is ovarian cancer. Alternatively, theendocrine-related cancer may be breast, uterine, endometrial, prostateor testicular cancer.

[0267] Any suitable technique for determining formation of the complexmay be used. For example, an antigen-binding molecule according to theinvention, having a reporter molecule associated therewith may beutilized in immunoassays. Such immunoassays include, but are not limitedto, radioimmunoassays (RIAs), enzyme-linked immunosorbent assays(ELISAS) and immunochromatographic techniques (ICTs), Western blottingwhich are well known those of skill in the art. For example, referencemay be made to “Current Protocols in Immunology” (1994, supra) whichdiscloses a variety of immunoassays that may be used in accordance withthe present invention. Immunoassays may include competitive assays asunderstood in the art or as for example described infra. It will beunderstood that the present invention encompasses qualitative andquantitative immunoassays.

[0268] Suitable immunoassay techniques are described for example in U.S.Pat. Nos. 4,016,043; 4,424,279; and 4,018,653. These include bothsingle-site and two-site assays of the non-competitive types, as well asthe traditional competitive binding assays. These assays also includedirect binding of a labeled antigen-binding molecule to a targetantigen.

[0269] Two site assays are particularly favored for use in the presentinvention. A number of variations of these assays exist all of which areintended to be encompassed by the present invention. Briefly, in atypical forward assay, an unlabelled antigen-binding molecule such as anunlabelled antibody is immobilized on a solid substrate and the sampleto be tested brought into contact with the bound molecule. After asuitable period of incubation, for a period of time sufficient to allowformation of an antibody-antigen complex, another antigen-bindingmolecule, suitably a second antibody specific to the antigen, labeledwith a reporter molecule capable of producing a detectable signal isthen added and incubated, allowing time sufficient for the formation ofanother complex of antibody-antigen-labeled antibody. Any unreactedmaterial is washed away and the presence of the antigen is determined byobservation of a signal produced by the reporter molecule. The resultsmay be either qualitative, by simple observation of the visible signal,or may be quantitated by comparing with a control sample containingknown amounts of antigen. Variations on the forward assay include asimultaneous assay, in which both sample and labeled antibody are addedsimultaneously to the bound antibody. These techniques are well known tothose skilled in the art, including minor variations as will be readilyapparent. In accordance with the present invention, the sample is onethat might contain an antigen including serum, whole blood, and plasmaor lymph fluid. The sample is, therefore, generally a circulatory samplecomprising circulatory fluid.

[0270] In the typical forward assay, a first antibody having specificityfor the antigen or antigenic parts thereof is either covalently orpassively bound to a solid surface. The solid surface is typically glassor a polymer, the most commonly used polymers being cellulose,polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene.The solid supports may be in the form of tubes, beads, discs ofmicroplates, or any other surface suitable for conducting animmunoassay. The binding processes are well known in the art andgenerally consist of cross-linking covalently binding or physicallyadsorbing, the polymer-antibody complex is washed in preparation for thetest sample. An aliquot of the sample to be tested is then added to thesolid phase complex and incubated for a period of time sufficient andunder suitable conditions to allow binding of any antigen present to theantibody. Following the incubation period, the antigen-antibody complexis washed and dried and incubated with a second antibody specific for aportion of the antigen. The second antibody has generally a reportermolecule associated therewith that is used to indicate the binding ofthe second antibody to the antigen. The amount of labeled antibody thatbinds, as determined by the associated reporter molecule, isproportional to the amount of antigen bound to the immobilized firstantibody.

[0271] An alternative method involves immobilizing the antigen in thebiological sample and then exposing the immobilized antigen to specificantibody that may or may not be labeled with a reporter molecule.Depending on the amount of target and the strength of the reportermolecule signal, a bound antigen may be detectable by direct labelingwith the antibody. Alternatively, a second labeled antibody, specific tothe first antibody is exposed to the target-first antibody complex toform a target-first antibody-second antibody tertiary complex. Thecomplex is detected by the signal emitted by the reporter molecule.

[0272] From the foregoing, it will be appreciated that the reportermolecule associated with the antigen-binding molecule may include thefollowing:

[0273] (a) direct attachment of the reporter molecule to theantigen-binding molecule;

[0274] (b) indirect attachment of the reporter molecule to theantigen-binding molecule; i.e., attachment of the reporter molecule toanother assay reagent which subsequently binds to the antigen-bindingmolecule; and

[0275] (c) attachment to a subsequent reaction product of theantigen-binding molecule.

[0276] The reporter molecule may be selected from a group including achromogen, a catalyst, an enzyme, a fluorochrome, a chemiluminescentmolecule, a lanthamide ion such as Europium (Eu34), a radioisotope and adirect visual label.

[0277] In the case of a direct visual label, use may be made of acolloidal metallic or non-metallic particle, a dye particle, an enzymeor a substrate, an organic polymer, a latex particle, a liposome, orother vesicle containing a signal producing substance and the like.

[0278] A large number of enzymes suitable for use as reporter moleculesis disclosed in U.S. Pat. Nos. 4,366,241; 4,843,000; and 4,849,338.Suitable enzymes useful in the present invention include alkalinephosphatase, horseradish peroxidase, luciferase, beta-galactosidase,glucose oxidase, lysozyme, malate dehydrogenase and the like. Theenzymes may be used alone or in combination with a second enzyme that isin solution.

[0279] Suitable fluorochromes include, but are not limited to,fluorescein isothiocyanate (FITC), tetramethylrhodamine isothiocyanate(TRITC), R-Phycoerythrin (RPE), and Texas Red. Other exemplaryfluorochromes include those discussed by Dower et al. (InternationalPublication WO 93/06121). Reference also may be made to thefluorochromes described in U.S. Pat. Nos. 5,573,909 (Singer et al.) and5,326,692 (Brinkley et al.). Alternatively, reference may be made to thefluorochromes described in U.S. Pat. Nos. 5,227,487; 5,274,113;5,405,975; 5,433,896; 5,442,045; 5,451,663; 5,453,517; 5,459,276;5,516,864; 5,648,270; and 5,723,218.

[0280] In the case of an enzyme immunoassay, an enzyme is conjugated tothe second antibody, generally by means of glutaraldehyde or periodate.As will be readily recognized, however, a wide variety of differentconjugation techniques exist which are readily available to the skilledartisan. The substrates to be used with the specific enzymes aregenerally chosen for the production of, upon hydrolysis by thecorresponding enzyme, a detectable color change. Examples of suitableenzymes include those described supra. It is also possible to employfluorogenic substrates, which yield a fluorescent product rather thanthe chromogenic substrates noted above. In all cases, the enzyme-labeledantibody is added to the first antibody-antigen complex, allowed tobind, and then the excess reagent washed away. A solution containing theappropriate substrate is then added to the complex ofantibody-antigen-antibody. The substrate will react with the enzymelinked to the second antibody, giving a qualitative visual signal, whichmay be further quantitated, usually spectrophotometrically, to give anindication of the amount of antigen which was present in the sample.

[0281] Alternately, fluorescent compounds, such as fluorescein,rhodamine and the lanthamide, europium (Eu), may be chemically coupledto antibodies without altering their binding capacity. When activated byillumination with light of a particular wavelength, thefluorochrome-labeled antibody adsorbs the light energy, inducing a stateto excitability in the molecule, followed by emission of the light at acharacteristic color visually detectable with a light microscope. Thefluorescent-labeled antibody is allowed to bind to the firstantibody-antigen complex. After washing off the unbound reagent, theremaining tertiary complex is then exposed to light of an appropriatewavelength. The fluorescence observed indicates the presence of theantigen of interest. Immunofluorometric assays (IFMA) are wellestablished in the art and are particularly useful for the presentmethod. However, other reporter molecules, such as radioisotope,chemiluminescent or bioluminescent molecules may also be employed.

[0282] In a particularly preferred embodiment, the condition fordiagnosis is ovarian cancer. In this instance, a combinationimmunoenzymemetric assay is preferably employed which makes use of anantigen-binding molecule as described for example in Section 4 togetherwith an antigen-binding molecule against an ovarian cancer antigen suchas CA125. For example, the CA125 or other antigen is immobilized on asolid support such as magnetic beads with a first antibody and then asecond antibody labeled with an enzyme is allowed to bind to the CA125or to the other antigen. After appropriate washing, the complex isincubated in the presence of a fluorogenic substrate. The amount ofenzyme-labeled antibody that binds to the solid support is directlyproportional to the concentration of CA125 or other antigen in the testsample. A standard curve may also be constructed and concentrations ofCA125 or other antigens may be determined in an unknown sample using thestandard curve. An exemplary protocol for performing this assay isdescribed, for example, by Robertson et al (1999, Clin. Chem.45:651-658).

[0283] Inhibin may be determined in a similar manner to CA125 or to theother antigen. Particularly useful assays include an (alpha)C IFMA, aPro(alpha)C ELISA or a RIA. For example, in a preferred embodiment, anantigen-binding molecule to the Pro region of the alpha subunit is usedto immobilize inhibin molecules containing this region to a solidsupport such as a microtiter plate, magnetic bead or other suitablesurface. A second antigen-binding molecule as described in Section 4 andlabeled with an enzyme such as alkaline phosphatase is used to detectbound inhibin. A similar assay is described in Groome et al., (1996,supra) with the exception that an antigen-binding molecule directed tothe carboxyl terminal end of the alpha subunit ((alpha)C) was usedinstead of an antigen-binding molecule according to the invention.

[0284] The antigen-binding molecules of the invention can also beapplied to the conventional (alpha)C IFMA. For example, theseantigen-binding molecules may be used for the capture antibody in placeof the caprylic acid/ammonium polyclonal antibody raised against humaninhibin (alpha)C subunit fusion protein (Forage et al., 1987, InInhibin: Non-Steroidal Regulation of Follicle Stimulating HormoneSecretion, Burger et al., Eds., Raven Press. Serono Symposium42:89-103). The subject antigen-binding molecules can also be used asthe reporter or labeled antigen-binding molecule in place of theimmunopurified sheep polyclonal antibody raised against human inhibin(alpha)C subunit fusion protein (Forage et al., 1987, supra; Robertsonet al., 1997, supra).

[0285] 6. Compositions

[0286] The invention also encompasses a composition for use in elicitingan immune response in a mammal which response includes production ofelements that specifically bind the (alpha)C portion of a mammalianinhibin alpha subunit, comprising an immuno-interactive fragment,variant or derivative as broadly described above (“immunogenic agents”),together with a pharmaceutically acceptable carrier. Optionally, saidcomposition further comprises an adjuvant.

[0287] A further feature of the invention is the use of theantigen-binding molecules of the invention (“therapeutic agents”) asactives, together with a pharmaceutically acceptable carrier, in acomposition for protecting or treating patients against a conditionassociated with aberrant concentrations of a mammalian inhibin in amammal.

[0288] Depending upon the particular route of administration, a varietyof pharmaceutically acceptable carriers, well known in the art, may beused. These carriers may be selected from sugars, starches, celluloseand its derivatives, malt, gelatin, talc, calcium sulfate, vegetableoils, synthetic oils, polyols, alginic acid, phosphate bufferedsolutions, emulsifiers, isotonic saline, and pyrogen-free water.

[0289] Any suitable route of administration may be employed forproviding a mammal or a patient with a composition of the invention. Forexample, oral, rectal, parenteral, sublingual, buccal, intravenous,intra-articular, intramuscular, intra-dermal, subcutaneous,inhalational, intraocular, intraperitoneal, intracerebroventricular,transdermal and the like may be employed. Intra-muscular andsubcutaneous injection is appropriate, for example, for administrationof immunogenic compositions, vaccines and DNA vaccines.

[0290] Dosage forms include tablets, dispersions, suspensions,injections, solutions, syrups, troches, capsules, suppositories,aerosols, transdermal patches and the like. These dosage forms may alsoinclude injecting or implanting controlled releasing devices designedspecifically for this purpose or other forms of implants modified to actadditionally in this fashion. Controlled release of an immunogenic or atherapeutic agent may be effected by coating the same, for example, withhydrophobic polymers including acrylic resins, waxes, higher aliphaticalcohols, polylactic and polyglycolic acids and certain cellulosederivatives such as hydroxypropylmethyl cellulose. In addition,controlled release may be effected by using other polymer matrices,liposomes and/or microspheres.

[0291] Compositions suitable for oral or parenteral administration maybe presented as discrete units such as capsules, sachets or tablets eachcontaining a pre-determined amount of one or more immunogenic agents ofthe invention, as a powder or granules or as a solution or a suspensionin an aqueous liquid, a non-aqueous liquid, an oil-in-water emulsion ora water-in-oil liquid emulsion. Such compositions may be prepared by anyof the methods of pharmacy but all methods include the step of bringinginto association one or more immunogenic agents as described above withthe carrier which constitutes one or more necessary ingredients. Ingeneral, the compositions are prepared by uniformly and intimatelyadmixing the immunogenic agents of the invention with liquid carriers orfinely divided solid carriers or both, and then, if necessary, shapingthe product into the desired presentation.

[0292] The above compositions may be administered in a manner compatiblewith the dosage formulation, and in such amount as is therapeuticallyeffective or immunogenically effective as the case may be. In thisregard, the dose of immunogenic agent administered to a mammal should besufficient to elicit an immune response that includes the production ofelements that specifically bind to the (alpha)C potion of a mammalianinhibin alpha-subunit.

[0293] Alternatively, the dose of therapeutic agent administered to apatient should be sufficient to effect a beneficial response in thepatient over time such as a reduction in the level of a mammalianinhibin or to ameliorate the condition to be treated. The quantity ofthe therapeutic agent(s) to be administered may depend on the subject tobe treated inclusive of the age, sex, weight and general healthcondition thereof. In this regard, precise amounts of the therapeuticagent(s) for administration will depend on the judgement of thepractitioner. In determining the effective amount of the therapeuticagent to be administered in the treatment or prophylaxis of thecondition associated with aberrant levels of a mammalian inhibin, thephysician may evaluate circulating plasma levels, progression of thecondition, and the production of anti-inhibin antibodies.

[0294] In any event, those of skill in the art may readily determinesuitable dosages of the immunogenic and therapeutic agents of theinvention. Such dosages may be in the order of nanograms to milligramsof the immunogenic agents of the invention.

[0295] An immunogenic agent according to the invention can be mixed,conjugated or fused with other antigens, including B or T cell epitopesof other antigens. In addition, it can be conjugated to a carrier asdescribed below.

[0296] When an haptenic peptide is used (i.e., a peptide which reactswith cognate antibodies, but cannot itself elicit an immune response),it can be conjugated with an immunogenic carrier. Useful carriers arewell known in the art and include for example: thyroglobulin; albuminssuch as human serum albumin; toxins, toxoids or any mutantcross-reactive material (CRM) of the toxin from tetanus, diphtheria,pertussis, Pseudomonas, E. coli, Staphylococcus, and Streptococcus;polyamino acids such as poly(lysine:glutamic acid); influenza; RotavirusVP6, Parvovirus VP1 and VP2; hepatitis B virus core protein; hepatitis Bvirus recombinant vaccine and the like. Alternatively, a fragment orepitope of a carrier protein or other immunogenic protein may be used.For example, an haptenic peptide can be coupled to a T cell epitope of abacterial toxin, toxoid or CRM. In this regard, reference may be made toU.S. Pat. No. 5,785,973.

[0297] The immunogenic compositions may include an adjuvant as is wellknown in the art. Suitable adjuvants include, but are not limited to:surface active substances such as bexadecylamine, octadecylamine,octadecyl amino acid esters, lysolecithin, dimethyldioctadecylammoniumbromide, N,N-dicoctadecyl-N′, N′bis(2-hydroxyethyl-propanediamine),methoxyhexadecylglycerol, and pluronic polyols; polyamines such aspyran, dextransulfate, poly IC carbopol; peptides such as muramyldipeptide and derivatives, dimethylglycine, tuftsin; oil emulsions; andmineral gels such as aluminum phosphate, aluminum hydroxide or alum;lymphokines, and QuilA.

[0298] In a further embodiment, a polynucleotide of the invention may beused as an immunogenic composition in the form of a “naked DNA”composition as is known in the art. For example, an expression vector ofthe invention may be introduced into a mammal, where it causesproduction of an immuno-interactive fragment according to the inventionin vivo, against which the host mounts an immune response as for exampledescribed in Barry et al. (1995, Nature 377:632-635).

[0299] 7. Detection Kits

[0300] The present invention also provides kits for the detection of amammalian inhibin in a biological sample. These will contain one or moreagents described above depending upon the nature of the test methodemployed. In this regard, the kits may include one or more of animmuno-interactive fragment, variant, derivative, or antigen-bindingmolecule according to the invention. The kits may also optionallyinclude appropriate reagents for detection of labels, positive andnegative controls, washing solutions, dilution buffers and the like.

[0301] In order that the invention may be readily understood and putinto practical effect, particular preferred embodiments will now bedescribed by way of the following non-limiting examples.

EXAMPLES Example 1

[0302] Inhibin Immunofluorometric Assay (IFMA)

[0303] This IFMA is used in the measurement of inhibin in serum fromwomen with ovarian cancer and is presented at this juncture as areference to the following procedures. The IFMA is a sandwich antibodyassay in 96-well microtiter plates. The capture antiserum is As #128 andthe labeled antiserum As #41, both raised in sheep to the alpha subunitof human inhibin. Sheep As #128 was also boosted with human recombinant30-kDa inhibin. The microtiter plates were coated with a caprylic acidIgG cut of antiserum #128. Inhibin standard and serum samples were addedand incubated for 2 hours at room temperature. A biotinylated antiserum(As #41) which has been immunopurified by absorption to a column ofbovine (alpha)C subunit fusion protein (as previously described,Robertson et al., 1997, supra) followed by elution with a glycine (pH2.5) buffer, was added to bind to the antibody-bound inhibin (2-hourincubation at room temperature). Fluorescently (Eu) labeled-streptavidinthat has a high affinity for biotin is added (30 minutes at roomtemperature) and the Eu-bound streptavidin is counted in a time-resolvedfluorimeter. The Eu measured is proportional to the inhibin bound by thetwo antisera.

Example 2

[0304] Studies Involving Inhibin Alpha Subunit Peptides

[0305] The human inhibin alpha subunit sequence can be divided intothree parts, Pro (amino acids 19-61), (alpha)N (62-232) and (alpha)C(233-366, see Mason et al., 1986, Biochem. Biophys. Res. Commun.135:957-964 for sequence data) based on the known presence ofproteolytic cleavage sites and known isolation of these parts frombiological samples. Since the (alpha)C subunit is common to the vastmajority of inhibin forms, it has been used as the antigen for producingantisera (#128 and #41) in sheep.

[0306] To identify the various epitopes, 31 overlapping peptides (14amino acids long) of the human (alpha)C subunit were synthesized byChiron Mimotopes, Clayton, Vic (the sequences are presented in Table 1)with an N-terminal biotin attached. These peptides were then tested fortheir interaction with As #41 and #128 in the following assays incomparison with the native inhibin molecule (human recombinant 30 kDainhibin (hr-inhibin)).

[0307] Assay 1: Solid Phase Assay

[0308] This assay is a broad screen of the binding of the 31biotinylated peptides to As #41 and #128 as recommended by ChironMimotopes.

[0309] Methods

[0310] The biotinylated peptides were initially bound tostreptavidin-coated 96-well plates (2 hours at room temperature),antiserum #41 or #128 was then added (2 hours room temperature) to bindto the peptides. Detection of antiserum binding was assessed by afurther incubation with an anti-ovine IgG serum labeled with the enzyme,horseradish peroxidase. The enzyme activity was assessed by conversionof a colorless substrate to a colored product that is detected in aspectrophotometer. The enzyme activity measured was proportional to theextent of the binding of the peptides.

[0311] Results

[0312] The results, as presented in FIG. 1, show that As #41 and As #128bind in general to 4 peptide regions designated Region I (peptides 3-7),II(11-15), III (16-23) and IV (27-33). Region II peptides were oflimited solubility and as such the results were treated with cautionparticularly at high peptide concentrations. Peptides from this regionshowed limited responses or no response in any of the assays.

[0313] Assay 2: RIA Using Antisera #41, #128 and Rabbit Antiserum #1989as Reference

[0314] This assay was used to determine which peptides compete withiodinated 30 kDa inhibin for As #41, #128 and #1989 (used in theoriginal inhibin RIA, Lapphorn et al., 1989, supra) in a RIA format.This assay identifies those peptides that bind to inhibin-bindingantibodies in the antiserum and provides a stricter affinity andspecificity assessment in identifying the appropriate epitopes than thatfound with Assay 1. The competition between peptide and hr-inhibin (usedas the standard) was assessed from their ED₅₀ values.

[0315] Methods

[0316] The RIA consisted of the competition of iodinated inhibin andeither hr-inhibin reference preparation, individual peptides or poolsfrom the 4 peptide regions, with As #41, #128 and in some instances, As#1989. The peptides/inhibin/iodinated inhibin and antisera (at aprescribed dilution) were incubated overnight at 4° C. using standardmethodologies. The antibody-bound iodinated inhibin wasimmunoprecipitated with the addition of an anti-sheep IgG serum raisedin goats and the radioactivity was measured in a gamma counter. Theconcentration of peptide that gives a 50% fall in binding of iodinatedinhibin (ED₅₀) was determined and this value was used to give a measureof the affinity of the peptide for the antiserum.

[0317] Results

[0318] The results presented in FIG. 2 and Table 2 indicate thatpeptides from Region 1, Region 3, and Region 4 show the lowest ED₅₀,indicating that these antisera bind peptides from these regions with thehighest affinity. As #41 showed a different range of affinities incomparison with As #128 with peptides #4-6 and #30-33 showing thehighest cross-reactions. As #128 showed cross-reaction with peptides#18-20 and #28-30. With regard to As #1989, the only peptide to showcompetition with hr-inhibin was peptide #30 (ED₅₀<0.01 mmole/mL) withthe others showing little or no evidence of competition (ED₅₀>2.5mmole/mL) (Table 2).

[0319] Assay 3: Competitive 2-Site Assay

[0320] This assay was developed to establish the relative importance ofthe individual peptides and peptide pools (identified as epitopes inAssays 1 and 2) in the sandwich antibody format used in the IFMA with As#128 as capture antibody and As #41 as detection antibody. Twoapproaches (a and b) were considered, Approach (a) explores thecross-reaction of peptides with inhibin for the immobilized As #128while Approach (b) assesses their cross-reaction with inhibin for As#41.

[0321] Approach (a). The biotinylated peptide pools and/or individualpeptides at various concentrations, in combination with a fixedconcentration of hr-inhibin, were incubated for 2 hours with As#128-coated plates. The plates were then washed to remove any unboundmaterial. Iodinated As #41 was then added to bind to the boundhr-inhibin, the plates washed and the bound radioactivity was measuredin a gamma counter. The counts measured were proportional to the amountof hr-inhibin bound to both antisera. The possibility that a peptidecontained two epitopes capable of linking As #128 and #41 was assessedin the absence of added hr-inhibin.

[0322] Results

[0323] The results, presented in FIG. 3, show that the binding of RegionI (FIG. 3a) and III (FIG. 3c) peptide pools and possibly Region IV (FIG.3d) peptide pools with As #128 partially competed (10-50%) withhr-inhibin while Region II pool showed no competition. The combinationof Region I-IV peptide pools totally suppressed binding (FIG. 3e).Individual peptides (peptide #5, #20 and #30, FIGS. 3f-h) showed asimilar range of binding to that seen with the corresponding Regionpeptide pools. These results suggest that the main epitopes on As #128are positioned around peptides #5, #20 and #30.

[0324] Approach (b). In this approach, the competition of peptides withthe As #41 antiserum using the sandwich antibody format was assessed.Hr-Inhibin was initially incubated with the As #128 coated wells for 1hour at room temperature. The biotinylated peptide pools and/orindividual peptides were incubated together with iodinated As #41 for 1hour and then added to the inhibin bound As #128 coated plates andincubated for 2 hours at room temperature. Plates were then washed toremove any unbound material and the resulting activity was measured inthe gamma counter.

[0325] Results

[0326] The results, presented in FIG. 4, showed that the binding ofRegion I (FIG. 4a) and IV (FIG. 4d) peptide pools to As #41 partiallyblocked the binding of As #41 to the inhibin-As#128 complex while thecombination of Regions I−IV or I+IV peptide pools totally suppressedbinding (FIG. 4e,f). Both Region pools II and III have limited effectsat high doses. Further data showed that individually, peptide #5 and #30each contributed 50% (FIG. 4g,i) while the combination of peptide #5 andeither peptide #29 (not shown) or #30 (FIG. 4j) competed totally withinhibin for As #41. These results indicate that the main epitopes on As#41 are located within peptides #5 and #29-30.

Example 3

[0327] Studies Utilizing Non-Biotinylated Peptide #5, #20 and #30

[0328] The above studies (Example 2) were undertaken using biotinylatedpeptides of approximate mass. The studies presented in Example 2identified 3 main peptides, #5, #20 and #30 as likely epitopes. A secondphase study was undertaken with these peptides synthesized in mg amountswith 95% purity by Chiron Mimotopes with anNH₂-Cys-Ser-Lys-Lys-Gly-amino terminal-spacer and a more precise massdetermination. The following studies were undertaken using these 3peptides.

[0329] RIA

[0330] This methodology consisted of determining the ED₅₀ of the peptidein the RIA using iodinated human recombinant inhibin as tracer witheither As #41, As #128 or As #1989 and graded doses of either hr-inhibinor peptide as described in Example 2.

[0331] As seen in Table 3, based on the ED₅₀ values, peptide #5cross-reacted strongly with hr-inhibin for As #41 while the otherpeptides were less reactive.

[0332] Two-Site Assay Design

[0333] The above RIA design is not directly comparable to the sandwichantibody design used in the IFMA as the RIA design is based oncompetition of peptide with inhibin. On the other hand, the IFMA designis based on the total amount of inhibin bound which is a combination ofboth antibody affinity and concentration. In order to establish to whatextent epitopes to these peptides contribute to the overall IFMA, thefollowing two-site assay designs were used.

[0334] (i) Peptides #5, #20 and #30 (0, 0.1-1 micromolar) were incubatedwith the As #128 coated plate for 1.5 hours at RT and the wells washed.The highest dose of peptide used was saturating.

[0335] (ii) Hr-Inhibin was then added as a saturating or near saturatingdose to the As #128 coated plate and incubated for 2 hours at roomtemperature and the plate washed.

[0336] (iii) Biotinylated As #41 was added to the above wells andincubated for 2 hours at RT. Plates were then washed and counted.

[0337] The contribution of each of the peptides to the overall bindingwas then assessed. As seen in Table 4 and FIG. 5, peptide #5 and #20showed a graded suppression in binding (65% and 23% respectively) withAs #128 while a combination of peptide #5 and #20 gave 83% suppression.Peptide #30 showed no suppression at all.

[0338] (iv) In a variation to the above design, biotinylated #41 waspre-incubated with peptide (0.1-1.0 micromolar) for 1.5 hours at roomtemperature and then added to the As #128 coated plate pre-bound withinhibin as for (i)-(iii). As seen in FIG. 6, peptide #5 showed 50%inhibition, while peptide #30 showed 25-30% but not saturating.Combination of peptide #5 and #30 led to 74% decrease. Peptide #20showed no inhibition.

[0339] As another approach, the competition of combinations of peptidesat a maximum saturating dose was assessed in the IFMA (FIGS. 7-9). Itcan be seen in FIG. 7 that the addition of peptides #5, #20 and #30 tothe IFMA resulted in an almost total suppression in binding whilepre-absorption with one peptide for example resulted in a lessersuppression. This suppression in binding provides a measure of thecontribution of that particular epitope to the assay.

[0340] The following conclusions were drawn from Table 5 and FIGS. 7-9.

[0341] (1) Peptides #5, #20 and #30 are responsible for the majority(95%) of inhibin binding in the (alpha)C IFMA. In relation to thecapture antibody (#128), peptide #5 is the most important althoughpeptide #20 does make a small contribution. This contribution is moreevident when the #5 peptide epitope is absorbed out with #5 peptide.Peptide #5 and peptide #30 regions are the primary epitopes on As #41.

[0342] (2) In an alternative sandwich antibody assay design where As#41was used as both as coating and labeled antibody, the addition ofpeptide #5 to both the coating and labeled antibodies in the absence ofinhibin resulted in significant binding (FIG. 8). These results suggestthat there are two epitopes on peptide #5 (termed epitopes 5a and 5b)and that the two antibodies in As #41 can bind this 14-amino acidresidue peptide simultaneously. It has not been established if the sameantibodies are present in As #128.

[0343] (3) Absorption of As #128 by peptides #5, #20 and #30 resulted inpartial suppression (55%) only indicating that there is another majorepitope in As #128 which has not been identified (FIG. 7c). Thisconclusion is despite the observation (FIG. 4) that the combination ofpeptides from all 4 regions resulted in total suppression. It is unclearwhere this epitope is located within the inhibin (alpha)C subunit.

[0344] (4) Why is it that peptides #20 and #30 show a low cross-reactionwith inhibin in the RIA yet show a relatively high contribution in theIFMA compared to peptide #5? One explanation is that compared withpeptide #5, epitopes #20 and #30 are present at high binding siteconcentrations although with low affinity that favors the IFM.

Example 4

[0345] Production of Mouse Anti-Inhibin (alpha)C Monoclonal Antibodies

[0346] Mouse monoclonal antibodies (designated PO# Mabs) were raisedagainst a recombinant inhibin (alpha)C subunit-beta galactosidase fusionprotein based on the hybridoma procedure as outlined in Groome et al.(1994, In “Inhibin and inhibin-related proteins,” Burger, Ed., Frontiersin Endocrinology, Vol. 3, Ares Serono Symposia). The hybridomas werescreened and cloned against both recombinant human inhibin A andpro-(alpha)C (a fragment of the alpha subunit of inhibin).

[0347] Characterization of Mabs

[0348] Antibody Affinity

[0349] The affinity of the various Mabs for inhibin based on ED₂₅ valueswas determined by radioimmunoassay according to assay 2 of Example 2using iodinated human inhibin A as tracer. Mabs (at an antibody dilutionto give 50% maximum iodinated inhibin binding, normally 1:500-1:2000dilutions of the original culture medium) were incubated with iodinatedhuman recombinant inhibin A in the presence of human recombinant inhibinA overnight at room temperature. The iodinated inhibin-antibody complexwas precipitated by an anti-mouse IgG serum and the radioactivitydetermined in a gamma counter.

[0350] Specificity

[0351] The binding of the 41 biotinylated peptides as set forth in Table7 (peptide set 2) to the mouse monoclonal antibodies (PO# series) wasassessed as follows:

[0352] a) Assay 1 (solid phase assay). The assay was undertaken asoutlined in assay 1 of Example 2. Biotinylated peptides (24micromoles/L) were initially bound to streptavidin-coated plates. Mab(at an appropriate dilution to give a detectable response, 1:1000,1:10000 dilution of the culture medium) was then added and incubated for1.5 hr. The amount of bound Mab was determined using horseradishperoxidase-bound anti-mouse IgG serum and enzyme activity detected at450-630 nm using an ELISA plate reader.

[0353] b) Assay 2 (radioimrnunoassay, RIA) using iodinated human inhibinA as tracer. Mabs (at an appropriate dilution, see above) were incubatedwith iodinated human recombinant inhibin in the presence of biotinylatedpeptides (0.8 and 0.08 micromoles/L final concentration) overnight atroom temperature.

[0354] Results

[0355] The affinity of the PO# Mabs as determined from RIA competitionstudies with inhibin is presented in Tables 8 and 9.

[0356] The specificity of the Mabs based on binding of the biotinylatedpeptides either in a solid phase binding assay or by RIA is alsopresented in Tables 8 and 9.

[0357] Discussion

[0358] As seen in Table 10, the Mabs, based on their specificity to thebiotinylated peptides, are directed to three epitopic regions seen withthe ovine polyclonal antisera mentioned above.

[0359] a) PO#6, PO#22 are immuno-interactive with peptides 2-7 of Set 2,which correspond to peptide #5 of Table 1.

[0360] b) PO#12, PO#14 are immuno-interactive with peptides 22-27 of Set2, which correspond to peptides #21-23 of Set 1. This region (i.e., theregion defined by peptides 22-27) appears to be distinct from epitope#20 (peptides #18-20 of Set 1) although there is potential overlap ofsequence. Perusal of FIGS. 1, 2 and Table 2 indicates that significantbinding with As #128 is present in this region of the inhibin sequence,although less than the nearby region corresponding to peptides #18-20(epitope #20). It is prudent to conclude that epitope #20 may comprisetwo epitopes, most likely #18-20 Set 1 (#19-21 Set 2, designated 20a)and #21-23 Set 1 (#22-27 Set 2, designated 20b) and that PO#12, PO#14are immuno-interactive with the latter.

[0361] c) Mabs PO#9, PO#19, PO#23, PO#25, PO#26 are immuno-interactivewith peptides 35-40 Set 2 or 30-32 Set 1. These Mabs are comparable withpeptide #30 shown in Table 1.

Example 5

[0362] Development of Alpha Subunit ELISAs

[0363] An ELISA system using 96-well microtiter plates was developedconsisting of one alpha subunit antibody as coating antibody and analkaline phosphatase-linked second antibody as label. The alkalinephosphatase activity was amplified using an ELISA amplification kit(Gibco, Life Technologies, Rockville Md., USA). The plate was initiallycoated with monoclonal antibody at 2 micrograms/well in 0.1 Mbicarbonate buffer pH 9.4 overnight at room temperature and blocked with50 mM TRIS/HCl, 1% bovine serum albumin (BSA) pH 7.4.

[0364] ELISA Procedure

[0365] The ELISAs in application to non-serum samples consisted of 100microliters sample or recombinant human (rh) inhibin A standard(provided by National Institute of Biological Standards and Control,Potters Bar, Herts, UK) in assay buffer (100 mM TRIS/HCl, 154 mM NaCl,5% Triton-X-100, 10% BSA pH, 7.5) and 100 microliters assay buffer. Inthe assay of serum, the inhibin A standard (100 microliters) was dilutedin assay buffer. Inhibin-free serum (100 microliters) was also added tomake a total well volume of 200 microliters. The inhibin-free serum wasobtained by incubating serum with an immobilized inhibin alpha subunitantibody. Repeated extractions resulted in no detectable inhibinimmunoactivity as determined by the alpha subunit ELISAs. Serum sampleswere initially boiled in the presence of SDS (2% final concentration)and diluted 1:1 with assay buffer before adding 100 microliters to thewells. The inhibin-free serum and the SDS boiling steps were included tooffset any potential matrix effects of serum known to affect otherinhibin ELISAs although the need for these specific steps had not beenassessed.

[0366] The plate was incubated with shaking overnight at roomtemperature. The wells were washed, and alkaline phosphatase (AP)-linkedantibody added, incubated with shaking for 3 hours at room temperatureand washed again. The substrate (NADPH, Gibco) was added and the plateincubated for 2 hours with shaking at room temperature. The amplifyingenzymes (alcohol dehydrogenase and diaphorase, Gibco) were added andincubated for 5-15 min until appropriate color had developed. The platewas read at 490/630 nm on an ELISA plate reader.

Example 6

[0367] Characteristics of the Inhibin Alpha ELISAs

[0368] Inhibin alpha ELISAs were developed using PO#14 and PO#23 Mabs ascoating antibody and AP-R1 as detection antibody. Serial dilutions ofstandard and serum or human follicular fluid gave parallel responses inthe various assays (FIGS. 10a, 10 b). The characteristics of theseassays are outlined in Table 11. The sensitivity of the ELISAs based oninhibin values calculated 2 standard deviations above the assay blankranged from 6-15 pg/mL serum. The levels of inhibin alpha in normal seraand human follicular fluid using these assays are presented in Table 13.

[0369] The specificity of the ELISAs was assessed by determining thecross-reaction of inhibin-related proteins in the various ELISAs. Asseen in Table 12, in comparison with the inhibin A standard, inhibin Band the alpha subunit fragment, Pro-(alpha)C, showed different degreesof cross-reaction in the various ELISAs.

[0370] As a result of an initial characterization of these ELISAs (seebelow), a combination of PO#14+PO#23 as coating antibodies and APlinked-R1 antibody as tracer was also assessed and its characteristicsare also presented in Table 11 and 12 and FIG. 10c.

Example 7

[0371] Specificity of the Inhibin Alpha ELISAs

[0372] It was unclear from the above characterization studies what wasthe specificity of the various ELISA assays in terms of their ability todetect inhibin alpha subunit monomer and (alpha)(beta) subunit dimers.To characterize further, IVF serum, male serum and serum from women withovarian granulosa cell tumours and mucinous tumours were fractionated bya combined immunoaffinity, Prep-PAGE/electroelution procedure similar tothat published previously by our group (Robertson et al., 1996, J. Clin.Endocrinol. Metab. 81:669-676, Robertson et al., 1997, J. Clin.Endocrinol. Metab. 82:889-896).

[0373] Inhibin forms were separated into their various molecular weightforms by this procedure and thus available for assessment by the variousinhibin assays. As seen in FIG. 11, a comparison of the molecular weightprofiles obtained with the 14-R1 and 23-R1 ELISAs for IVF serum and maleserum showed that the 14-R1 ELISA gave a molecular weight patternsimilar to that seen with the Pro-(alpha)C ELISA with 25-40 k inhibinforms primarily detected. In contrast, 23-R1 ELISA detected highmolecular weight forms in the 50-100 k range in greater abundance,similar to that seen with inhibin A and B ELISAs. These data suggestthat 14-R1 ELISA is directed more to the alpha subunit monomer while the23-R1 ELISA is directed more to the inhibin dimer.

[0374] Since the purpose of the proposed inhibin alpha ELISA was todetect all alpha subunit containing forms, i.e., both free alphasubunits and inhibin dimer, a further ELISA was devised consisting ofboth PO#14 and PO#23 as coating antibodies with AP-R1 as label, the aimof which was to combine the specificities of the 14-R1 and 23-R1 ELISAs.The characteristics of this ELISA are included in the various Tables andFigures considered for the individual ELISAs. The 14+23-R1 ELISA wasmore sensitive than the other inhibin alpha ELISAs with goodreproducibility (Table 11). The molecular weight patterns of inhibins inIVF and male serum determined by the 14+23-R1 ELISA is a mixture ofpatterns of both 14-R1 and 23-R1 assays (FIG. 11).

Example 8

[0375] Application to Serum from Women with Ovarian Cancers

[0376] The application of the various ELISAs to fractionated serum fromwomen with ovarian cancer showed a similar molecular weight pattern forall three ELISAs (FIG. 12) as different from that seen in IVF and maleserum, perhaps reflecting the high levels of monomeric alpha subunitforms compared to the dimeric forms present in these cancer samples.

[0377] The three ELISAs (14-R1, 23-R1, 14+23-R1) were then applied toserum from normal postmenopausal women (>55 years) and postmenopausalwomen with a range of ovarian cancers. As seen in Tables 14 and 15, incomparison with the IFMA, the three ELISAs readily detected inhibinlevels in granulosa and mucinous tumours compared to normal controlswith largely similar degrees of discrimination (Table 15). The 14+23-R1ELISA showed the largest difference between cancer and control groups.Regression analysis between serum inhibin levels determined by the IFMAand each of the inhibin alpha ELISAs showed good correlations (Table 16,FIG. 13). These data suggest that 14+23-R1 is marginally better than the14-R1 but based on the higher specificity of the 14+23-R1 ELISA for allinhibin forms, it is probably the better ovarian cancer assay.

Example 9

[0378] Other Applications of PO#14 and PO#23 Antisera

[0379] Other ELISAs

[0380] Current inhibin A and B and Pro-(alpha)C ELISAs use the R1 MAb asalpha subunit label (hereinafter referred to as the “Groome” assays andthe like). Studies were undertaken to replace the R1 MAb with either thePO#14 or PO#23 monoclonal antibody. Neither MAb in combination with the(beta)B subunit MAb (C5) gave a response in the ELISA. A comparison ofELISA assays in the fractionation of IVF and male serum showed that thecurrent Groome inhibin A ELISA (consisting of the (beta)A subunit MAb(E4) and R1) showed little differences with the other inhibin alphaELISAs, except that higher molecular weight forms (>80 k) were detectedwith the Groome inhibin A ELISA (FIG. 12). However, the combination ofINPRO MAb which detects the Pro-region of the alpha subunit with PO#14(INPRO-Ap-14) detected the presence of high molecular weight forms ofPro-(alpha)C not detected with the traditional Pro-R1 MAb combination.These findings suggest that the PO#14 MAb is a better MAb than R1 inconjunction with INPRO MAb in detecting Pro-(alpha)C forms. Acombination of PO#23 and INPRO resulted in an insensitive assay.

[0381] Immunocytochemistry

[0382] Previous studies by many groups have shown that the alpha subunitR1 antibody used as an immunocytochemical reagent readily detectsgranulosa cell tumours but not mucinous or other epithelial cellcancers. Studies using PO#14 and PO#23 as immunocytochemical reagentsshowed that these Mabs also detected granulosa cell tumours, but inaddition PO#14 readily detected a range of tumours including ovarianmucinous epithelial cancers. These studies suggest the PO#14 may beuseful in the immunocytochemical identification of mucinous tumours aswell as other ovarian cancers not currently possible with R1.

GENERAL CONCLUSIONS

[0383] Five (possibly 6) epitopes on the inhibin (alpha)C subunit havebeen identified (see Table 5) with epitopes #5a+5b representing 65-73%of the 30 kDa inhibin binding, epitope #20a and #20b, 13%, and epitope#30, 28%, of the mixture, although following the pre-absorption of the#5 epitopes, the others take on a larger role. There is an additionalepitope recognized by As #128 which has not been identified. It isunclear to what extent these various epitopes are important in thespecificity of the overall assay as it is likely that they maycontribute differently according to the type and form of inhibin beingdetected.

[0384] It would appear that an antibody to epitope 5a, which isrecognized by As #128, is sufficient to act as a capture antibody, whileepitopes #5b and #30 appear to be the key epitopes recognized by As #41.However it should be noted that there is a third epitope recognized byAs# 128 representing 45% of the total binding which has not as yet beenidentified.

[0385] Epitopes in the peptide #5 sequence are located near the aminoterminal of the (alpha)C subunit. Based on preliminary analysis usingthe peptides and the solid assay procedure presented in Assay 1, theseepitopes are probably different to that detected by the inhibin (alpha)Csubunit monoclonal antibody of Groome et al. (11994, Clin. Endocrinol.40:717-723) and used in the SEROTEC™ (alpha)(beta) dimer ELISA whichprimarily detects peptide 3 as listed in Table 1. However, theseepitopes in the peptide #5 sequence may be similar although notnecessarily identical to the sequences used in the alpha-alpha inhibinassay provided commercially by the company Medgenix. (See Table 5 andFIG. 7, see Robertson et al., 1996, J. Cell. Endocrinol. Metabol.81:669-676 for further details).

[0386] Peptides #20a, #20b and #30 as epitopes are unique. Peptide #30also shows a high affinity to rabbit antiserum #1989, which was employedin the earlier discussed inhibin RIA. Previous studies by other workers(Lambert-Messerlian et al., 1995, J. Cell. Endocrinol. Metabol. 80:3043)had localized the As #1989 epitope on the alpha-subunit to a differentregion (amino acid sequences 326-341 of the full alpha subunit or aminoacids 94-109 of the (alpha)C subunit region, See FIG. 9). However in thepresent study peptide #30 (from both the first and second series ofpeptides) was the only peptide to compete with inhibin for thisantiserum. We thus presume that the observations by Lambert-Messerlianand colleagues are incorrect.

[0387] An interesting observation from this study is that both peptide#5 and #30 show high sequence homology across a range of species (rat,bovine, ovine, human) with 13 of 14 amino acids of both peptides commonbetween the human and the other species. Thus an assay based on these(alpha)C subunit sequences would be appropriate in detecting inhibinalpha subunit in a range of species. Since the sequences of the (beta)Aand (beta)B subunits show little or no differences over a range ofspecies, combination of an antibody to the (beta)A/(beta)B subunitsequences and to one of these alpha-subunit peptide sequences wouldprovide a basis for an “all species” assays of inhibin A and B. At themoment the R1 alpha-subunit antibody used in the human inhibin A and BELISAs made by Groome show variable cross-reaction with inhibin fromother species. In fact Groome has produced specific antibodies to thealpha-subunit of bovine and ovine inhibin in order to detect inhibin Aand B in these species.

[0388] Three inhibin alpha ELISAs were developed to replace the IFMA asan ovarian cancer diagnostic. These assays exhibit differentspecificities for the various inhibin forms, however the ability ofthese assays to discriminate between controls and ovarian cancer wassimilar to that observed with the IFMA.

[0389] While the three inhibin alpha ELISAs are more sensitive than theIFMA it is unclear which assay is preferred as an ovarian cancer markerat this point. Further studies with a larger number of samples mayresolve this issue. Because of the differing sensitivities between the14-R1 and 23-R1 ELISAs, the combination assay 14+23-R1 ELISA wouldappear to be the most appropriate. It is worth noting that the 14+23-R1ELISA is more sensitive than the others as well as giving the largestdiscrimination (difference between control values and cancer values)relative to the other ELISAs.

[0390] PO#14 and PO#23 Mabs appear to be of value in detectingparticular forms of inhibin not detected by the presentinhibin/Pro-(alpha)C ELISAs and are likely to be useful in developingnew assays for these proteins.

[0391] PO#14 and PO#23 Mabs, and particularly PO#14 are considerablybetter than R1 in detecting various types of ovarian cancers byimmunocytochemistry. Thus PO#14 and PO#23 Mabs appear to be usefulreagents in detecting ovarian cancers by this technique.

[0392] All references, patents and patent applications referred toherein are incorporated herein by reference.

[0393] Throughout the specification, the aim has been to describe thepreferred embodiments of the invention without limiting the invention toany one embodiment or specific collection of features. Those of skill inthe art will therefore appreciate that, in light of the instantdisclosure, various modifications and changes can be made in theparticular embodiments exemplified without departing from the scope ofthe present invention. All such modifications and changes are intendedto be included within the scope of the appended claims. TABLE 1 Initialset of peptides derived from the human (alpha)C subunit examined in thisstudy Peptides 1 and 2 were Chiron quality control samples and notincluded in this study. Single letter code for the amino acids is used.A biotinylated 4 amino acid spacer (SGSG) with an N-terminal biotin isattached to the N- terminus of each peptide. Hydro=hydrophobicity index.Linker sequence is SEQ ID NO: 77. Peptide Position relative to # Linker+ Peptide Hydro Mol Wt SEQ ID NO SEQ ID NO: 2 1,2 quality controlpeptides 3 SGSG STPLMSWPWSPSAL 0.65 1830.07 3  1-14 4 SGSGMSWPWSPSALRLLQ 0.62 1942.24 4  5-18 5 SGSG WSPSALRLLQRPPE 0.38 1920.18 5 9-22 6 SGSG ALRLLQRPPEEPAA 0.26 1831.09 6 13-26 7 SGSG LQRPPEEPAAHANC0.18 1802.96 7 17-30 8 SGSG PEEPAAHANCHRVA 0.15 1771.90 8 21-34 9 SGSGAAHANCHRVALNIS 0.30 1746.92 9 25-38 10 SGSG NCHRVALNISFQEL 0.39 1914.1310 29-42 11 SGSG VALNISFQELGWER 0.42 1932.13 11 33-46 12 SGSGISFQELGWERWIVY 0.62 2096.34 12 37-50 13 SGSG ELGWERWIVYPPSF 0.61 2049.2913 41-54 14 SGSG ERWIVYPPSFIFHY 0.69 2124.41 14 45-58 15 SGSGVYPPSFIFHYCHGG 0.65 1894.11 15 49-62 16 SGSG SFIFHYCHGGCGLH 0.63 1848.0516 53-66 17 SGSG HYCHGGCGLHIPPN 0.48 1774.95 17 57-70 18 SGSGGGCGLHIPPNLSLP 0.56 1644.87 18 61-74 19 SGSG LHIPPNLSLPVPGA 0.60 1694.9619 65-78 20 SGSG PNLSLPVPGAPPTP 0.49 1626.85 20 69-82 21 SGSGLPVPGAPPTPAQPY 0.49 1674.90 21 73-86 22 SGSG GAPPTPAQPYSLLP 0.47 1678.8922 77-90 23 SGSG TPAQPYSLLPGAQP 0.42 1709.90 23 81-94 24 SGSGPYSLLPGAQPCCAA 0.57 1660.90 24 85-98 25 SGSG LPGAQPCCAALPGT 0.53 1568.8025  89-102 26 SGSG QPCCAALPGTMRPL 0.52 1728.06 26  93-106 27 SGSGAALPGTMRPLHVRT 0.36 1790.10 27  97-110 28 SGSG GTMRPLHVRTTSDG 0.161797.99 28 101-114 29 SGSG PLHVRTTSDGGYSF 0.28 1806.93 29 105-118 30SGSG RTTSDGGYSFKYET 0.05 1881.96 30 109-122 31 SGSG DGGYSFKYETVPNL 0.251859.98 31 113-126 32 SGSG SFKYETVPNLLTQH 0.34 1947.15 32 117-130 33SGSG ETVPNLLTQHCACI 0.54 1812.06 33 121-134

[0394] TABLE 2 ED₅₀ values for the 31 peptides obtained in the RIA withthe various antisera. As#41 As#128 As#1989 Peptide No. ED₅₀ nmoles/mLED₅₀ nmoles/mL ED₅₀ nmoles/mL 3 38 >2.5 4 1.3 3.8 >2.5 5 0.0000153.8 >2.5 6 0.026 1.8 >2.5 7 1.25 1.6 >2.5 8 >10 4.8 >2.5 9 >10 >10 >2.510 >10 >10 >2.5 11 >10 >10 >2.5 12 >10 >10 >2.5 13 >10 >10 >2.514 >10 >10 >2.5 15 >10 >10 >2.5 16 >10 >10 >2.5 17 1.05 10 >2.5 18 1.050.06 >2.5 19 2.1 <0.01 >2.5 20 3.6 0.02 >2.5 21 1 0.156 >2.5 22 50.156 >2.5 23 6.2 0.5 >2.5 24 6.2 0.9 >2.5 25 5 2 >2.5 26 1 0.625 >2.527 5 2 >2.5 28 4 0.625 >2.5 29 5 0.156 >2.5 30 2.5 0.6 <0.01 31 2.41.4 >2.5 32 0.29 2.5 >2.5 33 4 2.5 >2.5

[0395] TABLE 3 ED₅₀ values for hr-inhibin A as standard and peptides #5,#20 and #30 obtained in the RIA with the various antisera. ED₅₀ values(nmole/ml) As#41 As#128 As#1989 Hr-inhibin* 0.002-8 0.002 0.0001 Peptide#5 0.0004 >10 >10 Peptide #20 >5 0.15 >5 Peptide #30 >5 >5 2.5

[0396] TABLE 4 The effect of pre-immunoabsorption of As#128 and/or As#41with peptides #5, #20 and #30 in the inhibin IFMA. This data is derivedfrom FIG. 5 and is presented as the percentage inhibition of binding ofthe 2ng-inhibin dose by the 3 peptides, individually or combined. As#128as % suppression Coating Antibody As#41 as Label at 2ng inhibin Bufferbuffer 0 #5, #20, #30 #5, #20, #30 95 Buffer #5 67 #5 buffer 55 #5 #5 73#5 #30 80 #5, #20 buffer 55 #20 buffer 13 #20 #30 34 #20 #5 70 Buffer#30 28

[0397] TABLE 5 Summary of data for the 31 peptides in terms of theirrelative contributions in the various assays. +++++ majorcontribution, + minor contribution. Based on these data peptides #5, #20and #30 were chosen. Peptide #19 may be preferred in comparison withpeptide #20, however its solubility is limited based on itshydrophobicity index (see Table 1). #41 #41 2-site #128 #128 2-siteAntibody competitive combination Antibody #128 RIA competitive screen#41 RIA assay epitopes Peptide screen ED₅₀ assay #1989 Tube Assay 1Assay 2 Assay 3 Assay 3 No. Assay 1 Assay 2 Assay 3 RIA 3 3 +++ + ++ 4+++ ++ 4 + + + 5 +++ ++++++ ++++ 5 + + ++ 6 +++ +++ 6 + ++ + 7 +++ 7 +++ + 28 + ++ 18 + +++ + 29 +++ + +++ 100% 5 + 29 19 + +++ + 30 +++ +++++ 100% 5 + 30 20 + +++ + +++++ 31 + ++ ++ 28 ++ ++ ++ 32 +++ ++ 29 +++++ ++ 33 ++ + 30 ++ ++ ++ 31 ++ 32 ++ 33 ++

[0398] TABLE 6 Available inhibin alpha subunit antisera used inimmunoassays (alpha)C epitope (alpha)C subunit region subunit sequenceas (alpha)C Title Ref Assay Specificity Antiserum antigen subunit Groome1, 2 ELISAs Inhibin A, B, mouse  1-32 aa  1-32 aa (R1) Pro-(alpha)Cmonoclonal (R1) Medgenix 3 inhibin, inhibin polyclonal,  1-17 aa  1-17aa alpha subunit mouse 15-32 aa  15-32 monoclonal aa Monash 4 RIAinhibin, inhibin rabbit polyclonal bovine 31k 109-122 (#1989) alphasubunit (#1989) inhibin aa This study Monash 5 IFMA inhibin, inhibinsheep polyclonal (alpha)C subunit  9-22 aa alpha subunit (#128, #41)fusion protein This study (alpha)C subunit  69-82 fusion protein aa(alpha)C subunit 109-122 fusion protein aa

[0399] TABLE 7 Second set of peptides derived from the human (alpha)Csubunit examined in this study Set 1 peptides correspond to 30biotinylated peptides with 4 amino acid offset sequences of human(alpha)C subunit presented in Table 1. Set 2 peptides correspond to 41biotinylated peptides with 2-4 amino acid offset sequences. The commonsequence SGKG is a linker sequence. The presented Set 1 sequences areeither a combination of two Set 2 sequences (for example see peptide 3in Set 1 which is a combination of peptide 1 and 2 of Set 2) or amatching sequence with Set 1. linker Peptide Position relative Set SEQID SEQ ID to SEQ ID NO: Set 1 Offset 2 Offset NO: 77 Peptide Sequence NO2 3 4 1 2 SGKG STPLMSWPWSPSAL 34  1-14 3 2 2 SGKG PLMSWPWSPSALRL 35 3-16 4 4 3 2 SGKG MSWPWSPSALRLLQ 36  5-18 4 4 2 SGKG WPWSPSALRLLQRP 37 7-20 5 4 5 2 SGKG WSPSALRLLQRPPE 38  9-22 5 6 2 SGKG PSALRLLQRPPEEP 3911-24 6 4 7 4 SGKG ALRLLQRPPEEPAA 40 13-26 7 4 8 4 SGKG LQRPPEEPAAHANC41 17-30 8 4 9 4 SGKG PEEPAAHANCHRVA 42 21-34 9 4 10 4 SGKGAAHANCHRVALNIS 43 25-38 10 4 11 4 SGKG NCHRVALNISFQEL 44 29-42 11 4 12 4SGKG VALNISFQELGWER 45 33-46 12 4 13 4 SGKG ISFQELGWERWIVY 46 37-50 13 414 4 SGKG ELGWERWIVYPPSF 47 41-54 14 4 15 4 SGKG ERWIVYPPSFIFHY 48 45-5815 4 16 4 SGKG VYPPSFIFHYCHGG 49 49-62 16 4 17 4 SGKG SFIFHYCHGGCGLH 5053-66 17 4 18 4 SGKG HYCHGGCGLHIPPN 51 57-70 18 4 19 4 SGKGGGCGLHIPPNLSLP 52 61-74 19 4 20 4 SGKG LHIPPNLSLPVPGA 53 65-78 20 4 21 4SGKG PNLSLPVPGAPPTP 54 69-82 21 4 22 2 SGKG LPVPGAPPTPAQPY 55 73-86 2123 2 SGKG VPGAPPTPAQPYSL 56 75-88 22 4 24 2 SGKG GAPPTPAQPYSLLP 57 77-9022 25 2 SGKG PPTPAQPYSLLPGA 58 79-92 23 4 26 2 SGKG TPAQPYSLLPGAQP 5981-94 23 27 2 SGKG AQPYSLLPGAQPCC 60 83-96 24 4 28 2 SGKG PYSLLPGAQPCCAA61 85-98 24 29 4 SGKG SLLPGAQPCCAALP 62  87-100 25 4 30 4 SGKGLPGAQPCCAALPGT 63  89-102 26 4 31 4 SGKG QPCCAALPGTMRPL 64  93-106 27 432 4 SGKG AALPGTMRPLHVRT 65  97-110 28 4 33 4 SGKG GTMRPLHVRTTSDG 66101-114 29 4 34 4 SGKG PLHVRTTSDGGYSF 67 105-118 30 4 35 2 SGKGRTTSDGGYSFKYET 68 109-122 30 36 2 SGKG TSDGGYSFKYETVP 69 111-124 31 4 372 SGKG DGGYSFKYETVPNL 70 113-126 31 38 2 SGKG GYSFKYETVPNLLT 71 115-12832 4 39 2 SGKG SFKYETVPNLLTQH 72 117-130 32 40 2 SGKG KYETVPNLLTQHCA 73119-132 33 4 41 4 SGKG ETVPNLLTQHCACI 74 121-134

[0400] TABLE 8 Assessment of binding by solid phase binding andradioimmunoassay of the 41 biotinylated peptides to the various PPO#monoclonal antibodies R1 R1 PO-6 PO-6 PO-9 PO-9 PO-12 PO-12 PO-14 PO-14Set 2 Offset SBP RIA SBP RIA SBP RIA SBP RIA SBP RIA 1 2 2 2 +++ ΦΦΦ +++a) 3 2 +++ ΦΦΦ +++ 4 2 + ΦΦΦ +++ 5 2 + ΦΦΦ +++ 6 2 Φ 7 4 8 4 9 4 10 4 114 12 4 13 4 14 4 15 4 16 4 17 4 18 4 19 4 20 4 21 4 22 2 +++ Φ +++ Φ 232 +++ ΦΦΦ +++ ΦΦ 24 2 +++ ΦΦΦ +++ ΦΦΦ 25 2 +++ ΦΦΦ +++ ΦΦΦ 26 2 +++ ΦΦΦ+++ ΦΦΦ 27 2 ++ Φ +++ ΦΦ 28 2 29 4 30 4 31 4 32 4 33 4 34 4 35 2 +++ Φ36 2 +++ ΦΦΦ 37 2 ++ ΦΦΦ 38 2 + ΦΦΦ 39 2 (+) Φ 40 2 41 4

[0401] TABLE 9 PO-19 PO-19 PO-22 PO-22 PO-23 PO-23 PO-25 PO-25 PO-26PO-26 Set 2 offset SBP RIA SBP RIA SBP RIA SBP RIA SBP RIA 1 2 ΦΦ 2 2+++ ΦΦ 3 2 +++ ΦΦΦ 4 2 +++ ΦΦΦ 5 2 +++ ΦΦ 6 2 7 4 8 4 9 4 10 4 11 4 12 413 4 14 4 15 4 16 4 17 4 18 4 19 4 20 4 21 4 22 2 23 2 24 2 25 2 26 2 272 28 2 29 4 30 4 31 4 32 4 33 4 34 4 ΦΦ 35 2 +++ ΦΦΦ +++ ΦΦΦ +++ ΦΦΦ +++ΦΦΦ 36 2 +++ ΦΦΦ +++ ΦΦΦ +++ ΦΦΦ +++ ΦΦΦ 37 2 +++ ΦΦΦ ++ ΦΦΦ ++ ΦΦΦ ++ΦΦΦ 38 2 ++ ΦΦΦ ++ ΦΦΦ + ΦΦΦ + ΦΦΦ 39 2 + ΦΦ + Φ + Φ + Φ 40 2 Φ (+) Φ +Φ 41 4

[0402] TABLE 10 Summary of the affinity and inhibin peptide specificityof the Mabs from Table 7, 8 and 9. Biotinylated peptides Set 1 and Set 2are included for ease of comparison with the data in the provisionalpatent. A comparison with Groome R1 and #1989 antibodies is alsopresented. Epitope Epitope Affinity for region inhibin (ED25, (Peptideregion (Peptide Epitope Mab nmoles/L) Set 2) Set 1) designation PO#6 nottested, low  2-7  3-6 #5 PO#22 not tested, low  2-7  3-6 #5 Groome R11.6  2-7  3-6 PO#12 37 22-27 21-23 PO#14 14.8 22-27 21-23 PO#9 lowaffinity 35-40 30-32 #30 PO#19 12 35-40 30-32 #30 PO#23 5.5 35-40 30-32#30 PO#25 9.4 35-40 30-32 #30 PO#26 low affinity 35-40 30-32 #30 #19890.19 35 30

[0403] TABLE 11 Characteristics of the inhibin alpha ELISAs AssayCoating Labeled working sensitivity Between assay Antibody antibodyrange (pg/well) (pg/well) variation PO#14 R1 1.5-100 1.5  19% (n = 7)PO#23 R1 0.8-100 0.8 8.3% (n = 7) PO#14 + R1 0.6-100 0.6 7.3% (n = 7)PO#23

[0404] TABLE 12 Specificity of the inhibin alpha using MAb combinationsPO#14−R1, PO#23−R1 and PO#14 + PO#23−R1. Data is presented in relationto the recombinant human (rh) inhibin A standard (=100) Average of twoexperiments. 14−R1 23−R1 14 + 23−R1 Preparation ELISA ELSIA ELISArh-inhibin AWHO 91/624 100 100 100 rh-inhibin BR&D systems 320 253 138Pro-(alpha)COB standard* 98.5 41.5 38.5 rh-activin APHIMR preparation<0.2 <0.2 <0.2

[0405] TABLE 13 Levels of inhibin in human serum and human follicularfluid using the inhibin alpha ELISAs Inhibin concentration (pg/mL) 14−R1ELISA 23−R1 ELISA 14 + 23−R1 ELISA postmenopausal <1.5 <1.5 <1.5 serumfemale serum pool 1 14 28 22 female serum pool 2 44 98 77 female serumpool 3 228 292 237 male serum 100 64 46 human follicular 60800 5880046000 fluid

[0406] The three female pools were prepared from serum collected as partof an in vitro fertilization program and combined into the 3 pools basedon their serum estradiol levels (pool 1<1 moles/L, pool 2<2 mmoles/L,pool 3>2 mmoles/L) TABLE 14 Serum inhibin levels determined by variousinhibin alpha assays in normal postmenopausal women and postmenopausalwomen with ovarian cancers. Values are presented as geometric mean ± 2SD14-R1 23-R1 14 + 23-R1 RIA IFMA ELISA ELISA ELISA n (mU/mL) (pg/mL)(pg/mL) (pg/mL) (pg/mL) Normal 61 <122 51.0 1.57 0.88 0.72  15.8-164.70.71-3.49 0.39-1.97 0.23-2.32 GCT 7 1918 4320 113 229 165  109-33800 187-99700  1.15-11000   2.19-23400   1.68-16300  Mucinous 8 319 102015.6 15.1 20.9  22.5-4504   144-7286 0.45-535  0.39-583  0.91-477 Serous 15 116 112 1.83 1.44 0.97  44.1-305.5 15.1-833  0.45-7.460.22-9.66 0.10-9.54 Endometrioid 8 114 154 2.11 1.83 4.86  21-619 15.5-1520  0.24-18.7 0.16-21.5 0.40-59.1 Undifferentiated 8 83.4 74.71.4* 1.08 1.08 Clear cell 44.6-156   9.6-581  0.44-2.65 0.35-3.32

[0407] TABLE 15 Discrimination between ovarian cancer and control groupsusing a variety of serum inhibin alpha assays based on the number ofvalues detected above the upper 2SD of the control values 14-R1 23-R114 + 23-R1 RIA IFMA ELISA ELISA ELISA Level of 122 165 3.49 1.98 2.33discrimination mU/mL* pg/mL pg/mL pg/mL pg/mL Normal 2/61 5/61 3/61 5/61GCT 7/7 7/7 7/7 7/7 7/7 Mucinous 5/8 7/8 7/8 6/8 7/8 Serous 5/15 4/153/15 5/15 5/15 Endometrioid 3/8 3/8 1/8 4/8 3/8 Undifferentiated Clearcell 1/8 1/8 0/8 1/8 0/8

[0408] TABLE 16 Correlation coefficients for comparisons between seruminhibin levels determined by the various assays Correlation X axis Yaxis coefficient (r) number of cases RIA IFMA 0.824 46 IFMA 14-R1 ELISA0.902 46 IFMA 23-R1 ELISA 0.906 46 IFMA 14 + 23-R1 ELISA 0.934 46 14-R123-R1 ELISA 0.946 46 ELISA RIA 14 + 23-R1 ELISA 0.852 46

[0409] It will be appreciated by those skilled in the art that changescould be made to the embodiments described above without departing fromthe broad inventive concept thereof. It is understood, therefore, thatthis invention is not limited to the particular embodiments disclosed,but it is intended to cover modifications within the spirit and scope ofthe present invention as defined by the appended claims.

1 77 1 405 DNA Artificial Sequence alpha C fragment of human inhibin 1tca act ccc ctg atg tcc tgg cct tgg tct ccc tct gct ctg cgc ctg 48 SerThr Pro Leu Met Ser Trp Pro Trp Ser Pro Ser Ala Leu Arg Leu 1 5 10 15ctg cag agg cct ccg gag gaa ccg gct gcc cat gcc aac tgc cac aga 96 LeuGln Arg Pro Pro Glu Glu Pro Ala Ala His Ala Asn Cys His Arg 20 25 30 gtagca ctg aac atc tcc ttc cag gag ctg ggc tgg gaa cgg tgg atc 144 Val AlaLeu Asn Ile Ser Phe Gln Glu Leu Gly Trp Glu Arg Trp Ile 35 40 45 gtg taccct ccc agt ttc atc ttc cac tac tgt cat ggt ggt tgt ggg 192 Val Tyr ProPro Ser Phe Ile Phe His Tyr Cys His Gly Gly Cys Gly 50 55 60 ctg cac atccca cca aac ctg tcc ctt cca gtc cct ggg gct ccc cct 240 Leu His Ile ProPro Asn Leu Ser Leu Pro Val Pro Gly Ala Pro Pro 65 70 75 80 acc cca gcccag ccc tac tcc ttg ctg cca ggg gcc cag ccc tgc tgt 288 Thr Pro Ala GlnPro Tyr Ser Leu Leu Pro Gly Ala Gln Pro Cys Cys 85 90 95 gct gct ctc ccaggg acc atg agg ccc cta cat gtc cgc acc acc tcg 336 Ala Ala Leu Pro GlyThr Met Arg Pro Leu His Val Arg Thr Thr Ser 100 105 110 gat gga ggt tactct ttc aag tat gag aca gtg ccc aac ctt ctc acg 384 Asp Gly Gly Tyr SerPhe Lys Tyr Glu Thr Val Pro Asn Leu Leu Thr 115 120 125 cag cac tgt gcttgt atc taa 405 Gln His Cys Ala Cys Ile 130 2 134 PRT ArtificialSequence alpha C fragment of human inhibin 2 Ser Thr Pro Leu Met Ser TrpPro Trp Ser Pro Ser Ala Leu Arg Leu 1 5 10 15 Leu Gln Arg Pro Pro GluGlu Pro Ala Ala His Ala Asn Cys His Arg 20 25 30 Val Ala Leu Asn Ile SerPhe Gln Glu Leu Gly Trp Glu Arg Trp Ile 35 40 45 Val Tyr Pro Pro Ser PheIle Phe His Tyr Cys His Gly Gly Cys Gly 50 55 60 Leu His Ile Pro Pro AsnLeu Ser Leu Pro Val Pro Gly Ala Pro Pro 65 70 75 80 Thr Pro Ala Gln ProTyr Ser Leu Leu Pro Gly Ala Gln Pro Cys Cys 85 90 95 Ala Ala Leu Pro GlyThr Met Arg Pro Leu His Val Arg Thr Thr Ser 100 105 110 Asp Gly Gly TyrSer Phe Lys Tyr Glu Thr Val Pro Asn Leu Leu Thr 115 120 125 Gln His CysAla Cys Ile 130 3 14 PRT Artificial Sequence inhibin alpha C amino acidsequence corresponding to peptide 3 of TABLE 1 3 Ser Thr Pro Leu Met SerTrp Pro Trp Ser Pro Ser Ala Leu 1 5 10 4 14 PRT Artificial Sequenceinhibin alpha C amino acid sequence corresponding to peptide 4 of TABLE1 4 Met Ser Trp Pro Trp Ser Pro Ser Ala Leu Arg Leu Leu Gln 1 5 10 5 14PRT Artificial Sequence inhibin alpha C amino acid sequencecorresponding to peptide 5 of TABLE 1 5 Trp Ser Pro Ser Ala Leu Arg LeuLeu Gln Arg Pro Pro Glu 1 5 10 6 14 PRT Artificial Sequence inhibinalpha C amino acid sequence corresponding to peptide 6 of TABLE 1 6 AlaLeu Arg Leu Leu Gln Arg Pro Pro Glu Glu Pro Ala Ala 1 5 10 7 14 PRTArtificial Sequence inhibin alpha C amino acid sequence corresponding topeptide 7 of TABLE 1 7 Leu Gln Arg Pro Pro Glu Glu Pro Ala Ala His AlaAsn Cys 1 5 10 8 14 PRT Artificial Sequence inhibin alpha C amino acidsequence corresponding to peptide 8 of TABLE 1 8 Pro Glu Glu Pro Ala AlaHis Ala Asn Cys His Arg Val Ala 1 5 10 9 14 PRT Artificial Sequenceinhibin alpha C amino acid sequence corresponding to peptide 9 of TABLE1 9 Ala Ala His Ala Asn Cys His Arg Val Ala Leu Asn Ile Ser 1 5 10 10 14PRT Artificial Sequence inhibin alpha C amino acid sequencecorresponding to peptide 10 o f TABLE 1 10 Asn Cys His Arg Val Ala LeuAsn Ile Ser Phe Gln Glu Leu 1 5 10 11 14 PRT Artificial Sequence inhibinalpha C amino acid sequence corresponding to peptide 11 o f TABLE 1 11Val Ala Leu Asn Ile Ser Phe Gln Glu Leu Gly Trp Glu Arg 1 5 10 12 14 PRTArtificial Sequence inhibin alpha C amino acid sequence corresponding topeptide 12 o f TABLE 1 12 Ile Ser Phe Gln Glu Leu Gly Trp Glu Arg TrpIle Val Tyr 1 5 10 13 14 PRT Artificial Sequence inhibin alpha C aminoacid sequence corresponding to peptide 13 o f TABLE 1 13 Glu Leu Gly TrpGlu Arg Trp Ile Val Tyr Pro Pro Ser Phe 1 5 10 14 14 PRT ArtificialSequence inhibin alpha C amino acid sequence corresponding to peptide 14o f TABLE 1 14 Glu Arg Trp Ile Val Tyr Pro Pro Ser Phe Ile Phe His Tyr 15 10 15 14 PRT Artificial Sequence inhibin alpha C amino acid sequencecorresponding to peptide 15 o f TABLE 1 15 Val Tyr Pro Pro Ser Phe IlePhe His Tyr Cys His Gly Gly 1 5 10 16 14 PRT Artificial Sequence inhibinalpha C amino acid sequence corresponding to peptide 16 o f TABLE 1 16Ser Phe Ile Phe His Tyr Cys His Gly Gly Cys Gly Leu His 1 5 10 17 14 PRTArtificial Sequence inhibin alpha C amino acid sequence corresponding topeptide 17 o f TABLE 1 17 His Tyr Cys His Gly Gly Cys Gly Leu His IlePro Pro Asn 1 5 10 18 14 PRT Artificial Sequence inhibin alpha C aminoacid sequence corresponding to peptide 18 o f TABLE 1 18 Gly Gly Cys GlyLeu His Ile Pro Pro Asn Leu Ser Leu Pro 1 5 10 19 14 PRT ArtificialSequence inhibin alpha C amino acid sequence corresponding to peptide 19o f TABLE 1 19 Leu His Ile Pro Pro Asn Leu Ser Leu Pro Val Pro Gly Ala 15 10 20 14 PRT Artificial Sequence inhibin alpha C amino acid sequencecorresponding to peptide 20 o f TABLE 1 20 Pro Asn Leu Ser Leu Pro ValPro Gly Ala Pro Pro Thr Pro 1 5 10 21 14 PRT Artificial Sequence inhibinalpha C amino acid sequence corresponding to peptide 21 o f TABLE 1 21Leu Pro Val Pro Gly Ala Pro Pro Thr Pro Ala Gln Pro Tyr 1 5 10 22 14 PRTArtificial Sequence inhibin alpha C amino acid sequence corresponding topeptide 22 o f TABLE 1 22 Gly Ala Pro Pro Thr Pro Ala Gln Pro Tyr SerLeu Leu Pro 1 5 10 23 14 PRT Artificial Sequence inhibin alpha C aminoacid sequence corresponding to peptide 23 o f TABLE 1 23 Thr Pro Ala GlnPro Tyr Ser Leu Leu Pro Gly Ala Gln Pro 1 5 10 24 14 PRT ArtificialSequence inhibin alpha C amino acid sequence corresponding to peptide 24o f TABLE 1 24 Pro Tyr Ser Leu Leu Pro Gly Ala Gln Pro Cys Cys Ala Ala 15 10 25 14 PRT Artificial Sequence inhibin alpha C amino acid sequencecorresponding to peptide 25 o f TABLE 1 25 Leu Pro Gly Ala Gln Pro CysCys Ala Ala Leu Pro Gly Thr 1 5 10 26 14 PRT Artificial Sequence inhibinalpha C amino acid sequence corresponding to peptide 26 o f TABLE 1 26Gln Pro Cys Cys Ala Ala Leu Pro Gly Thr Met Arg Pro Leu 1 5 10 27 14 PRTArtificial Sequence inhibin alpha C amino acid sequence corresponding topeptide 27 o f TABLE 1 27 Ala Ala Leu Pro Gly Thr Met Arg Pro Leu HisVal Arg Thr 1 5 10 28 14 PRT Artificial Sequence inhibin alpha C aminoacid sequence corresponding to peptide 28 o f TABLE 1 28 Gly Thr Met ArgPro Leu His Val Arg Thr Thr Ser Asp Gly 1 5 10 29 14 PRT ArtificialSequence inhibin alpha C amino acid sequence corresponding to peptide 29o f TABLE 1 29 Pro Leu His Val Arg Thr Thr Ser Asp Gly Gly Tyr Ser Phe 15 10 30 14 PRT Artificial Sequence inhibin alpha C amino acid sequencecorresponding to peptide 30 o f TABLE 1 30 Arg Thr Thr Ser Asp Gly GlyTyr Ser Phe Lys Tyr Glu Thr 1 5 10 31 14 PRT Artificial Sequence inhibinalpha C amino acid sequence corresponding to peptide 31 o f TABLE 1 31Asp Gly Gly Tyr Ser Phe Lys Tyr Glu Thr Val Pro Asn Leu 1 5 10 32 14 PRTArtificial Sequence inhibin alpha C amino acid sequence corresponding topeptide 32 o f TABLE 1 32 Ser Phe Lys Tyr Glu Thr Val Pro Asn Leu LeuThr Gln His 1 5 10 33 14 PRT Artificial Sequence inhibin alpha C aminoacid sequence corresponding to peptide 33 o f TABLE 1 33 Glu Thr Val ProAsn Leu Leu Thr Gln His Cys Ala Cys Ile 1 5 10 34 14 PRT ArtificialSequence inhibin alpha C amino acid sequence corresponding to peptide 1of TABLE 7 34 Ser Thr Pro Leu Met Ser Trp Pro Trp Ser Pro Ser Ala Leu 15 10 35 14 PRT Artificial Sequence inhibin alpha C amino acid sequencecorresponding to peptide 2 of TABLE 7 35 Pro Leu Met Ser Trp Pro Trp SerPro Ser Ala Leu Arg Leu 1 5 10 36 14 PRT Artificial Sequence inhibinalpha C amino acid sequence corresponding to peptide 3 of TABLE 7 36 MetSer Trp Pro Trp Ser Pro Ser Ala Leu Arg Leu Leu Gln 1 5 10 37 14 PRTArtificial Sequence inhibin alpha C amino acid sequence corresponding topeptide 4 of TABLE 7 37 Met Pro Trp Ser Pro Ser Ala Leu Arg Leu Leu GlnArg Pro 1 5 10 38 14 PRT Artificial Sequence inhibin alpha C amino acidsequence corresponding to peptide 5 of TABLE 7 38 Trp Ser Pro Ser AlaLeu Arg Leu Leu Gln Arg Pro Pro Glu 1 5 10 39 14 PRT Artificial Sequenceinhibin alpha C amino acid sequence corresponding to peptide 6 of TABLE7 39 Pro Ser Ala Leu Arg Leu Leu Gln Arg Pro Pro Glu Glu Pro 1 5 10 4014 PRT Artificial Sequence inhibin alpha C amino acid sequencecorresponding to peptide 7 of TABLE 7 40 Ala Leu Arg Leu Leu Gln Arg ProPro Glu Glu Pro Ala Ala 1 5 10 41 14 PRT Artificial Sequence inhibinalpha C amino acid sequence corresponding to peptide 8 of TABLE 7 41 LeuGln Arg Pro Pro Glu Glu Pro Ala Ala His Ala Asn Cys 1 5 10 42 14 PRTArtificial Sequence inhibin alpha C amino acid sequence corresponding topeptide 9 of TABLE 7 42 Pro Glu Glu Pro Ala Ala His Ala Asn Cys His ArgVal Ala 1 5 10 43 14 PRT Artificial Sequence inhibin alpha C amino acidsequence corresponding to peptide 10 o f TABLE 7 43 Ala Ala His Ala AsnCys His Arg Val Ala Leu Asn Ile Ser 1 5 10 44 14 PRT Artificial Sequenceinhibin alpha C amino acid sequence corresponding to peptide 11 o fTABLE 7 44 Asn Cys His Arg Val Ala Leu Asn Ile Ser Phe Gln Glu Leu 1 510 45 14 PRT Artificial Sequence inhibin alpha C amino acid sequencecorresponding to peptide 12 o f TABLE 7 45 Val Ala Leu Asn Ile Ser PheGln Glu Leu Gly Trp Glu Arg 1 5 10 46 14 PRT Artificial Sequence inhibinalpha C amino acid sequence corresponding to peptide 13 o f TABLE 7 46Ile Ser Phe Gln Glu Leu Gly Trp Glu Arg Trp Ile Val Tyr 1 5 10 47 14 PRTArtificial Sequence inhibin alpha C amino acid sequence corresponding topeptide 14 o f TABLE 7 47 Glu Leu Gly Trp Glu Arg Trp Ile Val Tyr ProPro Ser Phe 1 5 10 48 14 PRT Artificial Sequence inhibin alpha C aminoacid sequence corresponding to peptide 15 o f TABLE 7 48 Glu Arg Trp IleVal Tyr Pro Pro Ser Phe Ile Phe His Tyr 1 5 10 49 14 PRT ArtificialSequence inhibin alpha C amino acid sequence corresponding to peptide 16o f TABLE 7 49 Val Tyr Pro Pro Ser Phe Ile Phe His Tyr Cys His Gly Gly 15 10 50 14 PRT Artificial Sequence inhibin alpha C amino acid sequencecorresponding to peptide 17 o f TABLE 7 50 Ser Phe Ile Phe His Tyr CysHis Gly Gly Cys Gly Leu His 1 5 10 51 14 PRT Artificial Sequence inhibinalpha C amino acid sequence corresponding to peptide 18 o f TABLE 7 51His Tyr Cys His Gly Gly Cys Gly Leu His Ile Pro Pro Asn 1 5 10 52 14 PRTArtificial Sequence inhibin alpha C amino acid sequence corresponding topeptide 19 o f TABLE 7 52 Gly Gly Cys Gly Leu His Ile Pro Pro Asn LeuSer Leu Pro 1 5 10 53 14 PRT Artificial Sequence inhibin alpha C aminoacid sequence corresponding to peptide 20 o f TABLE 7 53 Leu His Ile ProPro Asn Leu Ser Leu Pro Val Pro Gly Ala 1 5 10 54 14 PRT ArtificialSequence inhibin alpha C amino acid sequence corresponding to peptide 21o f TABLE 7 54 Pro Asn Leu Ser Leu Pro Val Pro Gly Ala Pro Pro Thr Pro 15 10 55 14 PRT Artificial Sequence inhibin alpha C amino acid sequencecorresponding to peptide 22 o f TABLE 7 55 Leu Pro Val Pro Gly Ala ProPro Thr Pro Ala Gln Pro Tyr 1 5 10 56 14 PRT Artificial Sequence inhibinalpha C amino acid sequence corresponding to peptide 23 o f TABLE 7 56Val Pro Gly Ala Pro Pro Thr Pro Ala Gln Pro Tyr Ser Leu 1 5 10 57 14 PRTArtificial Sequence inhibin alpha C amino acid sequence corresponding topeptide 24 o f TABLE 7 57 Gly Ala Pro Pro Thr Pro Ala Gln Pro Tyr SerLeu Leu Pro 1 5 10 58 14 PRT Artificial Sequence inhibin alpha C aminoacid sequence corresponding to peptide 25 o f TABLE 7 58 Pro Pro Thr ProAla Gln Pro Tyr Ser Leu Leu Pro Gly Ala 1 5 10 59 14 PRT ArtificialSequence inhibin alpha C amino acid sequence corresponding to peptide 26o f TABLE 7 59 Thr Pro Ala Gln Pro Tyr Ser Leu Leu Pro Gly Ala Gln Pro 15 10 60 14 PRT Artificial Sequence inhibin alpha C amino acid sequencecorresponding to peptide 27 o f TABLE 7 60 Ala Gln Pro Tyr Ser Leu LeuPro Gly Ala Gln Pro Cys Cys 1 5 10 61 14 PRT Artificial Sequence inhibinalpha C amino acid sequence corresponding to peptide 28 o f TABLE 7 61Pro Tyr Ser Leu Leu Pro Gly Ala Gln Pro Cys Cys Ala Ala 1 5 10 62 14 PRTArtificial Sequence inhibin alpha C amino acid sequence corresponding topeptide 29 o f TABLE 7 62 Ser Leu Leu Pro Gly Ala Gln Pro Cys Cys AlaAla Leu Pro 1 5 10 63 14 PRT Artificial Sequence inhibin alpha C aminoacid sequence corresponding to peptide 30 o f TABLE 7 63 Leu Pro Gly AlaGln Pro Cys Cys Ala Ala Leu Pro Gly Thr 1 5 10 64 14 PRT ArtificialSequence inhibin alpha C amino acid sequence corresponding to peptide 31o f TABLE 7 64 Gln Pro Cys Cys Ala Ala Leu Pro Gly Thr Met Arg Pro Leu 15 10 65 14 PRT Artificial Sequence inhibin alpha C amino acid sequencecorresponding to peptide 32 o f TABLE 7 65 Ala Ala Leu Pro Gly Thr MetArg Pro Leu His Val Arg Thr 1 5 10 66 14 PRT Artificial Sequence inhibinalpha C amino acid sequence corresponding to peptide 33 o f TABLE 7 66Gly Thr Met Arg Pro Leu His Val Arg Thr Thr Ser Asp Gly 1 5 10 67 14 PRTArtificial Sequence inhibin alpha C amino acid sequence corresponding topeptide 34 o f TABLE 7 67 Pro Leu His Val Arg Thr Thr Ser Asp Gly GlyTyr Ser Phe 1 5 10 68 14 PRT Artificial Sequence inhibin alpha C aminoacid sequence corresponding to peptide 35 o f TABLE 7 68 Arg Thr Thr SerAsp Gly Gly Tyr Ser Phe Lys Tyr Glu Thr 1 5 10 69 14 PRT ArtificialSequence inhibin alpha C amino acid sequence corresponding to peptide 36o f TABLE 7 69 Thr Ser Asp Gly Gly Tyr Ser Phe Lys Tyr Glu Thr Val Pro 15 10 70 14 PRT Artificial Sequence inhibin alpha C amino acid sequencecorresponding to peptide 37 o f TABLE 7 70 Asp Gly Gly Tyr Ser Phe LysTyr Glu Thr Val Pro Asn Leu 1 5 10 71 14 PRT Artificial Sequence inhibinalpha C amino acid sequence corresponding to peptide 38 o f TABLE 7 71Gly Tyr Ser Phe Lys Tyr Glu Thr Val Pro Asn Leu Leu Thr 1 5 10 72 14 PRTArtificial Sequence inhibin alpha C amino acid sequence corresponding topeptide 39 o f TABLE 7 72 Ser Phe Lys Tyr Glu Thr Val Pro Asn Leu LeuThr Gln His 1 5 10 73 14 PRT Artificial Sequence inhibin alpha C aminoacid sequence corresponding to peptide 40 o f TABLE 7 73 Lys Tyr Glu ThrVal Pro Asn Leu Leu Thr Gln His Cys Ala 1 5 10 74 14 PRT ArtificialSequence inhibin alpha C amino acid sequence corresponding to peptide 41o f TABLE 7 74 Glu Thr Val Pro Asn Leu Leu Thr Gln His Cys Ala Cys Ile 15 10 75 75 000 76 4 PRT Artificial Sequence Linker Sequence 76 Ser GlySer Gly 1 77 4 PRT Artificial Sequence linker sequence 77 Ser Gly SerGly 1

We claim:
 1. An antigen-binding molecule that binds specifically to animmuno-interactive fragment that is interactive with an ovine polyclonalantibody selected from the group consisting of As #41 and As #128described by Robertson et al. (1996, J. Clin. Endocrinol. Metab.81:669-676) and As #1989 described by Lapphorn et al. (1989, New EnglandJ. Med. 321:790-793), with the proviso that said antigen-bindingmolecule is other than a member selected from the group consisting of apolyclonal antibody and the R1 monoclonal antibody described by Groomeet al (1993, J. Immunol. Meth. 165:167-176; 1994, Clin. Endocrinol.40:717-723).
 2. The antigen-binding molecule of claim 1, wherein theimmuno-interactive fragment comprises a sequence selected from any oneor more of SEQ ID NOs: 3, 4, 5, 6, 18, 19, 20, 21, 22, 23, 30, 31, 32,35, 36, 37, 38, 39, 40, 55, 56, 57, 58, 59, 60, 68, 69, 70, 71, 72 and73.
 3. The antigen-binding molecule of claim 1, wherein theimmuno-interactive fragment comprises a sequence selected from any oneor more of SEQ ID NOs: 5, 35, 36, 37, 38, 39 and
 40. 4. Theantigen-binding molecule of claim 1, wherein the immuno-interactivefragment comprises a sequence selected from any one or more of SEQ IDNOs: 18, 19, 20, 21, 22, 23, 31, 32, 55, 56, 57, 58, 59 and
 60. 5. Theantigen-binding molecule of claim 1, wherein the immuno-interactivefragment comprises a sequence selected from any one or more of SEQ IDNOs: 68, 69, 70, 71, 72 and
 73. 6. An immuno-interactive fragment of the(alpha)C portion of a mammalian inhibin alpha subunit, wherein saidfragment is interactive with an ovine polyclonal antibody selected fromthe group consisting of As #41 and As #128 described by Robertson et al.(1996, J. Clin. Endocrinol. Metab. 81:669-676) and As #1989 described byLapphorn et al. (1989, New England J. Med. 321:790-793).
 7. A method ofproducing a variant of an immuno-interactive fragment of the (alpha)Cportion of a mammalian inhibin alpha subunit, wherein said fragment isinteractive with an ovine polyclonal antibody selected from the groupconsisting of As #41 and As #128 described by Robertson et al. (1996, J.Clin. Endocrinol. Metab. 81:669-676) and As #1989 described by Lapphornet al. (1989, New England J. Med. 321:790-793), said method comprising:(a) combining a polypeptide whose sequence is distinguished from theimmuno-interactive fragment by substitution, deletion and/or addition ofat least one amino acid with at least one antigen-binding molecule thatbinds to said immuno-interactive fragment; and (b) detecting thepresence of a conjugate comprising said polypeptide and saidantigen-binding molecule, which indicates that said polypeptide is saidvariant.
 8. A method of producing an antigen-binding molecule that bindsspecifically to an immuno-interactive fragment of the (alpha)C portionof a mammalian inhibin alpha subunit, wherein said fragment isinteractive with an ovine polyclonal antibody selected from the groupconsisting of As #41 and As #128 described by Robertson et al. (1996, J.Clin. Endocrinol. Metab. 81:669-676) and As #1989 described by Lapphornet al. (1989, New England J. Med. 321:790-793), said method comprising:(a) producing an antigen-binding molecule against inhibin (alpha)C orfragment thereof; (b) combining the antigen-binding molecule with saidimmuno-interactive fragment, variant or derivative; and (c) detectingthe presence of a conjugate comprising said antigen-binding molecule andsaid fragment.
 9. An antigen-binding molecule produced by the method ofclaim
 8. 10. A composition for use in eliciting an immune response in amammal which response includes production of elements that specificallybind the (alpha)C portion of a mammalian inhibin alpha subunit, saidcomposition comprising an immuno-interactive fragment of the (alpha)Cportion of a mammalian inhibin alpha subunit, wherein said fragment isinteractive with an ovine polyclonal antibody selected from the groupconsisting of As #41 and As #128 described by Robertson et al. (1996, J.Clin. Endocrinol. Metab. 81:669-676) and As #1989 described by Lapphornet al. (1989, New England J. Med. 321:790-793), together with apharmaceutically acceptable carrier.
 11. A method for eliciting animmune response in a mammal which response includes production ofelements that specifically bind the (alpha)C portion of a mammalianinhibin alpha subunit, comprising administering to said mammal animmunogenically effective amount of a composition comprising animmuno-interactive fragment of the (alpha)C portion of a mammalianinhibin alpha subunit, wherein said fragment is interactive with anovine polyclonal antibody selected from the group consisting of As #41and As #128 described by Robertson et al. (1996, J. Clin. Endocrinol.Metab. 81:669-676) and As #1989 described by Lapphorn et al. (1989, NewEngland J. Med. 321:790-793), together with a pharmaceuticallyacceptable carrier.
 12. An isolated polynucleotide encodingimmuno-interactive fragment of the (alpha)C portion of a mammalianinhibin alpha subunit, wherein said fragment is interactive with anovine polyclonal antibody selected from the group consisting of As #41and As #128 described by Robertson et al. (1996, J. Clin. Endocrinol.Metab. 81:669-676) and As #1989 described by Lapphorn et al. (1989, NewEngland J. Med. 321:790-793).
 13. An expression vector comprising apolynucleotide encoding immuno-interactive fragment of the (alpha)Cportion of a mammalian inhibin alpha subunit, wherein said fragment isinteractive with an ovine polyclonal antibody selected from the groupconsisting of As #41 and As #128 described by Robertson et al. (1996, J.Clin. Endocrinol. Metab. 81:669-676) and As #1989 described by Lapphornet al. (1989, New England J. Med. 321:790-793), wherein thepolynucleotide is operably linked to a regulatory polynucleotide.
 14. Ahost cell containing the expression vector of claim
 13. 15. A method ofdetecting a mammalian inhibin in a biological sample suspected ofcontaining it, comprising: (a) contacting the biological sample with anantigen-binding molecule that binds specifically to animmuno-interactive fragment that is interactive with an ovine polyclonalantibody selected from the group consisting of As #41 and As #128described by Robertson et al. (1996, J. Clin. Endocrinol. Metab.81:669-676) and As #1989 described by Lapphorn et al. (1989, New EnglandJ. Med. 321:790-793), with the proviso that said antigen-bindingmolecule is other than a member selected from the group consisting of apolyclonal antibody and the R1 monoclonal antibody described by Groomeet al (1993, J. Immunol. Methods 165:167-176; 1994, Clin. Endocrinol.40:717-723); and (b) detecting the presence of a complex comprising thesaid antigen-binding molecule and the mammalian inhibin in saidcontacted sample.
 16. A method of diagnosing a condition associated withan aberrant concentration of a mammalian inhibin in a biological sampleof a patient, comprising: (a) contacting the biological sample with anantigen-binding molecule that binds specifically to animmuno-interactive fragment that is interactive with an ovine polyclonalantibody selected from the group consisting of As #41 and As #128described by Robertson et al. (1996, J. Clin. Endocrinol. Metab.81:669-676) and As #1989 described by Lapphorn et al. (1989, New EnglandJ. Med. 321:790-793), with the proviso that said antigen-bindingmolecule is other than a member selected from the group consisting of apolyclonal antibody and the R1 monoclonal antibody described by Groomeet al (1993, J. Immunol. Meth. 165:167-176; 1994, Clin. Endocrinol.40:717-723); (b) measuring the concentration of a complex comprisingsaid antigen-binding molecule and said mammalian inhibin in saidcontacted sample; and (c) relating said measured complex concentrationto the concentration of mammalian inhibin in said sample, wherein thepresence of said aberrant concentration is indicative of said condition.17. A method of diagnosing a condition associated with an aberrantconcentration of a mammalian inhibin and an aberrant concentration ofanother antigen in a biological sample of a patient, comprising: (a)contacting a biological sample of the patient with a firstantigen-binding molecule that binds specifically to animmuno-interactive fragment that is interactive with an ovine polyclonalantibody selected from the group consisting of As #41 and As #128described by Robertson et al. (1996, J. Clin. Endocrinol. Metab.81:669-676) and As #1989 described by Lapphorn et al. (1989, New EnglandJ. Med. 321:790-793), with the proviso that said first antigen-bindingmolecule is other than a member selected from the group consisting of apolyclonal antibody and the R1 monoclonal antibody described by Groomeet al (1993, J. Immunol. Meth. 165:167-176; 1994, Clin. Endocrinol.40:717-723); (b) contacting said biological sample or another biologicalsample obtained from said patient with a second antigen-binding moleculethat is immuno-interactive with said other antigen; (c) measuring theconcentration of a first complex comprising the first antigen-bindingmolecule and the mammalian inhibin in said contacted sample; (d)measuring the concentration of a second complex comprising the secondantigen-binding molecule and the other antigen in said contacted sample;and (e) relating said measured complex concentrations to theconcentration of mammalian inhibin and the concentration of the otherantigen in said sample, wherein the presence of said aberrantconcentrations is indicative of said condition.
 18. A method fortreating or preventing a condition associated with an aberrantconcentration of a mammalian inhibin in a mammal, comprisingadministering to said mammal a therapeutically effective amount of acomposition comprising an immuno-interactive fragment of the (alpha)Cportion of a mammalian inhibin alpha subunit, wherein said fragment isinteractive with an ovine polyclonal antibody selected from the groupconsisting of As #41 and As #128 described by Robertson et al. (1996, J.Clin. Endocrinol. Metab. 81:669-676) and As #1989 described by Lapphornet al. (1989, New England J. Med. 321:790-793), together with apharmaceutically acceptable carrier.
 19. A polypeptide that induces ananti-inhibin alpha subunit immune response when the polypeptide isadministered to a mammal, wherein the amino acid sequence of thepolypeptide comprises at least 6 contiguous residues of animmuno-interactive sequence selected from the group consisting of SEQ IDNOs: 3-6, 18-23, 30-32, 35-40, 55-60, and 68-73.
 20. The polypeptide ofclaim 19, wherein the polypeptide comprises at least 8 contiguousresidues of the immuno-interactive sequence.
 21. The polypeptide ofclaim 19, wherein the polypeptide comprises at least 10 contiguousresidues of the immuno-interactive sequence.
 22. The polypeptide ofclaim 19, wherein the polypeptide comprises at least 15 contiguousresidues of the immuno-interactive sequence.
 23. The polypeptide ofclaim 19, wherein the polypeptide comprises at least 20 contiguousresidues of the immuno-interactive sequence.
 24. The polypeptide ofclaim 19, wherein the polypeptide comprises at least 30 contiguousresidues of the immuno-interactive sequence.
 25. The polypeptide ofclaim 18, wherein the polypeptide comprises from 6 to 20 amino acidresidues.
 26. The polypeptide of claim 18, wherein the polypeptidecomprises from 10 to 15 amino acid residues.
 27. The polypeptide ofclaim 18, wherein the sequence of the polypeptide further comprises acarrier sequence linked to the immuno-interactive sequence.
 28. Thepolypeptide of claim 27, wherein the carrier sequence is selected fromthe group consisting of the sequence of keyhole limpet hemocyanin andthe sequence of bovine serum albumin.
 29. The polypeptide of claim 28,wherein the sequence of the polypeptide further comprises a linkersequence interposed between the carrier sequence and theimmuno-interactive sequence.
 30. The polypeptide of claim 29, whereinthe linker sequence is selected from the group consisting of SEQ ID NOs:76 and 77.